Protein kinase domain of the large subunit of herpes simplex type 2 ribonucleotide reductase (icp10pk) has anti-apoptotic activity

ABSTRACT

The invention relates to a method of treating neuronal apoptosis in a mammal using nucleic acid encoding HSV-2 ICP10PK, or a polypeptide encoded thereby. The invention further relates to a method of treating neuronal apoptosis in a mammal using ICP10PK in combination with a nucleic acid encoding bcl-2, or the polypeptide encoded thereby. The invention also relates to the use of ICP10PK and ICP10PK in combination with bcl-2 to treat non-neuronal diseases characterized by apoptosis.

BACKGROUND OF THE INVENTION

[0001] The lack of effective treatment for a number of neurologicaldiseases, many of which are fatal, is a significant public healthconcern. Approximately four million Americans have Alzheimer's disease(AD) and the number will increase to approximately fourteen million bythe year 2005, unless a cure or prevention is found. Similarly,amyotrophic lateral sclerosis (ALS), commonly known as Lou Gherig'sdisease, is a fatal neurodegenerative disease that affects approximately2 per 100,000 people. In the U.S. alone, more than 5,000 people arediagnosed with ALS each year. Approximately 15 million people in theU.S. suffer from diabetes, and most are at risk for development ofneuropathy associated with diabetes. The incidence of diabeticneuropathy is considered to be 6 in 10,000 people. Down syndrome (DS),which occurs in 1 of 1,000 live births, also causes severe developmentalbrain abnormalities. Moreover, DS patients develop AD by their fifthdecade of life. The common denominator for neurodegenerative disorders(which also include Parkinson's disease (PD) and Huntington disease(HD)) is selective neuronal loss due to programmed cell death orapoptosis. To date, there are no known cures or effective treatments forthese diseases. Acute neurological diseases involving apoptotic deathoffer a window of opportunity for short-term treatment. They includestroke, trauma and hypoxia-ischemia, where there is a delayed“secondary” brain injury in a “penumbra zone”, that is initially sparedand surrounds the area of most severe damage. Cell death in thesepenumbra areas is due to apoptosis and is amenable to anti-apoptotictherapeutic strategies that minimize brain damage. While stroke is thethird common cause of death in the US, it is a leading cause oflong-term disability. Over 400,000 subjects suffer from a first ischemicstroke each year in the US. Effective treatment is not currentlyavailable, and gene therapy is limited by the paucity of genes withanti-apoptotic activity in the CNS.

[0002] Following spinal cord injury (SCI), loss of motor neurons occursby mechanical tissue disruption as well as necrosis. Secondarily,degeneration results from a cascade of events triggered by the injuryand results in the activation of endogenous apoptosis, cell death.Apoptosis does not occur immediately after injuty; rather it occurs overa prolonged period of several weeks. The cause of the apoptosis includesloss of trophic support, intracellular oxidative stress incltdin2oxidative damage to, and activation of caspases including caspase-q andcaspase-3 in both neurons and microglia. Moreover an increase in theexpression of pro-apoptotic peptide Bax and a reciprocal decrease in theanti-apoptotic Bcl-2 peptide in the mitochondrial-enriched membranecompartments occurs with SCI. Such a loss of motor neurons causesparalysis and death.

[0003] Apoptosis is a normal physiological process observed in many celltypes and enables useful pruning of “mismatched” or excessive cellsduring development and maturation. Apoptosis is critical to modeling ofthe nervous system during embryonic development and in the regulationand function of the immune system. However, cellular homeostasis isdependent on a proper balance between survival/proliferation andapoptotic processes. Thus, excessive apoptosis can lead tonon-physiological death, and ultimately to disease states. In the adultnervous system, where there is little cell production and little celldeath, excessive apoptosis results in neurological diseases includingAD, ALS, DS, PD, and HID, or is subsequent to physical ischemic orchemical injury of the central nervous system (CNS). What exactlyprompts apoptosis in chronic disorders is unclear, but several stimuliare thought to play an etiologic role. Such stimuli include oxidativestress that may increase with age, loss of neurotrophic support,accumulated burden of endogenous and exogenous factors, and excessiverelease of neurotransmitters known as excitotoxins.

[0004] Features associated with apoptosis include cell shrinkage,exposure of phosphatidylserine on the outer surface of the membrane,plasma membrane blebbing, mitochondrial dysfunction, DNA cleavage,chromatin condensation, and formation of membrane bound apoptoticbodies. Apoptosis involves three phases: (i) a time-variable phasecalled commitment which refers to the time from the reception ofapoptotic stimuli to the irreversible initiation of the second phase,(ii) execution phase in which all of the dramatic changes associatedwith cell death occur, and (iii) clearance, which involves engulfment ofapoptotic bodies by neighboring cells or be professional phagocyteswithout the stimulation of an inflammatory response. The commitmentphase is influenced by the proliferative status of the cell, cell type,apoptotic stimulus and expression of regulatory genes that inhibit orpromote apoptosis. During the commitment phase, a cell is faced withmultile decisioii/chectk points. The nature and intensity of theilnCOMillng stimulus may detet-iiie whether the cell suLvives (if thedefense mechanisms can overcome the insult) or conmiits to undergo aseries of apoptotic phases. This death is characterized by severalmorphological changes including condensation of the cell nucleus andmembrane blebbing. In addition, there is DNA fragmentation caused by agroup of caspases (cysteine aspartases) that are specifically activatedin apoptotic cells by proteolytic cleavage. Indeed, the executionprocess of apoptosis occurs mainly via activation of a series ofproteolytic enzymes, termed caspases. Once active, caspases cleave anumber of cellular proteins including proteins involved in DNA repairand replication such as poly (ADP-ribose) polymerase (PARP),DNA-dependent protein kinase (DNA-PK), and Inhibitors ofCaspase-Activated DNAse (ICAD), as well as structural proteins, therebyinducing endonuclease activation and the characteristic morphologicalchanges associated with apoptosis.

[0005] Caspase-3 is one of the key executioners of apoptosis. Itsactivation requires proteolytic cleavage of the inactive procaspase-3into activated 17-20 kDa and 12 kDa subunits. Activated caspase-3 is, inturn, responsible, either partially or totally, for the proteolyticcleavage of many key proteins, such as PARP, that is involved in DNArepair. PARP cleavage is a crucial event in the commitment to undergoapoptosis. Again, cell homeostasis depends on the balance betweenapoptotic and survival/proliferation processes. Survival stimuli causethe membrane bound G protein, Ras, to adopt an active, GTP-bound state,and it, in turn, coordinates the activation of a multitude of downstreameffectors. The mitogenic/survival Ras/MEK/MAPK pathway begins with theactivation of Raf kinase and is followed by the activation of MAP kinase(MEK) and mitogen activated protein kinase (MAPK). A variety of genes,including those required for cell cycle progression, are targets forMAPK. The Ras/MEK/MAPK pathway is also involved in the control ofapoptosis, presumably by upregulating anti-apoptotic proteins such asbcl-2 or mcl-1 (Boonni, et. al., 1999, Science, 286:1358-1362).

[0006] Herpes Simplex Virus Type 2 (HSV-2) is a dual tropic virus thatinfects cells of the mucosal epithelium as well as cells of the nervoussystem. HSV-2 encodes a ribonucleotide reductase (RR) enzyme comprisedof two subunits, referred to as the large and small subunits, encoded bythe UL39 and UL40 genes, respectively. Within the large submit of HSV_2RR (ICP10), resides a protein kinase domain termed ICP10PK whosesequence is known in the art. ICP10PK exhibits intrinsic protein kinaseactivity and has previously been shown to cause neoplastictransformation in a variety of cell types.

[0007] Viruses depend on cells for their replication and they candifferenitially affect various signaling pathways. Herpes Simplex VirusType 1 (HSV-1) and HSV-2 can trigger or counteract apoptosis in acell-specific manner (Aubert et al., 1999, J. Virol., 73:2803-2813;Aubert et al., 1999, J. Virol., 73:10359-10370; Chou et al., 1992, Proc.Natl. Acad. Sci. USA, 89:3266-3270). Anti-apoptotic activity wasascribed to the HSV-1 and HSV-2 gene US3, and to the HSV-1 genes γ₁34.5,US5, ICP27, and LAT (Aubert et al., 1999, J. Virol., 73:2803-2813; Chouet al., 1992, Proc. Natl. Acad. Sci. USA, 89:3266-3270). However, theirexact mechanism of action and their activity in hippocampal neurons, ifany, are still poorly understood.

[0008] There is a long felt need for treatment programs which arrest oralleviate neurological diseases where the diseases are caused, at leastin part, by apoptosis of neuronal cells. The present invention satisfiesthis need.

BRIEF SUMMARY OF THE INVENTION

[0009] The invention includes a method of inhibiting neuronal apoptosisin a mammal. The method comprises administering to the mammal anapoptosis-inhibiting amount of an isolated nucleic acid encoding ICP10,or any mutant, variant, homolog, or fragment thereof havinganti-apoptotic activity.

[0010] In a preferred embodiment of the invention, neuronal apoptosis isassociated with a neurodegenerative disorder. In a further preferredembodiment, the neurodegenerative disorder is Alzheimer's disease (AD),amyotrophic lateral sclerosis (ALS), Down syndrome (DS), diabeticneuropathy, Parkinson's disease (PD), or Huntington disease (HD).

[0011] In another preferred embodiment, neuronal apoptosis is associatedwith an injury of the central or peripheral nervous system. In a furtherpreferred embodiment, the injury is the result of stroke, cerebralischemia, or chemical and/or physical trauma.

[0012] The invention also includes a method of inhibiting neuronalapoptosis in a mammal. The method comprises administering to the mammalan apoptosis-inhibiting inhibiting amount of the combination of anisolated nucleic acid encoding ICP10, or any mutant, variant, homolog,or fragment thereof having anti-apoptotic activity, and an isolatednucleic acid encoding bcl-2, or any mutant, variant, liomolog, orfragment thereof having anti-apoptotic activity.

[0013] In a preferred embodiment, the isolated nucleic acid encodingICP10 and the isolated nucleic acid encoding bcl-2 are polycistronic.

[0014] The invention also includes a method of inhibiting neuronalapoptosis in a mammal. The method comprises administering to the mammalan apoptosis-inhibiting amount of a vector comprising a nucleic acidencoding ICP10, or any mutant, variant, homolog, or fragment thereofhaving anti-apoptotic activity.

[0015] In a preferred embodiment, the vector is a virus or a plasmid. Ina further preferred embodiment, the virus is a herpesvirus, adenovirus,adeno associated virus, retrovirus, vaccinia virus, or canary pox virus.In yet a further preferred embodiment, the herpesvirus is HSV-2. Inanother embodiment, the HSV-2 comprises a mutation that renders theHSV-2 replication-defective. Further preferred, the mutation eliminatesthe ribonucleotide reductase domain of ICP10. In still another preferredembodiment, the ribonucleotide reductase domain is replaced with nucleicacid selected from the group consisting of a nucleic acid encoding LacZand a nucleic acid encoding bcl-2, or any mutant, variant, homolog, orfragment thereof having anti-apoptotic activity.

[0016] The invention includes a method of inhibiting neuronal apoptosisin a mammal. The method comprises administering to the mammal anapoptosis-inhibiting amount of a polypeptide encoded by a nucleic acidencoding ICP10, or any mutant, variant, homolog, or fragment thereofhaving anti-apoptotic activity.

[0017] In a preferred embodiment of the invention, the ICP10 polypeptideis fused to a polypeptide encoded by a nucleic acid encoding bcl-2, orany mutant, variant, homolog, or fragment thereof having anti-apoptoticactivity.

[0018] In another preferred embodiment of the invention, neuronalapoptosis is associated with a neurodegenerative disorder. Furtherpreferred, the neurodegenerative disorder is Alzheimer's disease (AD),amyotrophic lateral sclerosis (ALS), Down syndrome (DS), diabeticneuropathy, Parkinson's disease (PD), or Huntington disease (HD).

[0019] In another preferred embodiment, neuronal apoptosis is associatedwith an injury of the central or peripheral nervous system. In a furtherpreferred embodiment, the injury is the result of stroke, cerebralischemia, or chemical and/or physical trauma.

[0020] The invention includes a method of inhibiting neuronal apoptosisin a mammal. The method comprises administering to the mammal anapoptosis-inhibiting amount of the combination of a polypeptide encodedby a nucleic acid encoding ICP10, or any mutant, variant, homolog, orfragment thereof having apoptotic activity and a polypeptide encoded bya nucleic acid encoding bcl-2, or any mutant, variant, homolog, orfragment thereof having apoptotic activity.

[0021] The invention also includes a method of inhibiting apoptosis in amammal. The method comprises administering to the mammal anapoptosis-inhibiting amount of an isolated nucleic acid encoding ICP10,or any mutant, variant, homolog, or fragment thereof havinganti-apoptotic activity.

[0022] The invention includes a method of inhibiting apoptosis in amammal, wherein the method comprises administering to the mammal anapoptosis-inhibiting amount of the combination of an isolated nucleicacid encoding ICP10, or any mutant, variant, homolog, or fragmentthereof having anti-apoptotic activity, and an isolated nucleic acidencoding bcl-2, or any mutant, variant, homolog, or fragment thereofhaving anti-apoptotic activity.

[0023] The invention also includes a method of inhibiting apoptosis in amammal, wherein the method comprises administering to the mammal anapoptosis-inhibiting amount of a vector comprising a nucleic acidencoding ICP10, or any mutant, variant, homolog, or fragment thereofhaving anti-apoptotic activity.

[0024] The invention includes a method of inhibiting apoptosis in amammal, wherein the method comprising administering to the mammal anapoptosis-inhibiting amount of a polypeptide encoded by a nucleic acidencoding ICP10, or any mutant, variant, homolog, or fragment thereofhaving anti-apoptotic activity.

[0025] The invention also includes a method of inhibiting apoptosis in amammal, wherein the method comprises administering to the mammal anapoptosis-inhibiting amount of a polypeptide encoded by a nucleic acidencoding ICP10, or any mutant, variant, homolog, or fragment thereofhaving anti-apoptotic activity.

[0026] In a preferred embodiment of the invention, the polypeptide isfused to a polypeptide encoded by a nucleic acid encoding bcl-2, or anymutant, variant, homolog, or fragment thereof having anti-apoptoticactivity.

[0027] The invention includes a composition comprising a fusionpolypeptide, vherein1 the fuisio polypeptide comprises a portion ofICP10 polypeptide and a portion of a bcl-2 polypeptide. The compositionfiuther comprises a pharmaceutically acceptable canrier tlicrefo(i.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The foregoing summary, as well as the following detaileddescription of preferred embodiments of the invention, will be betterunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there is shown in the drawingsembodiments which are presently preferred. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities shown.

[0029] In the drawings:

[0030]FIG. 1A is a graph depicting the apoptotic index of HEK293, JHL15,and JHLA1 cells treated with staurosporine (STS) and D-mannitol(D-Mann).

[0031]FIG. 1B through 1D are images of photomicrographs depicting thenuclear morphology of TUNEL-stained STS-treated HEK293, JHL15, and JHLA1cells, respectively.

[0032]FIG. 2 is an image of an agarose gel depicting inhibition of DNAfragmentation by ICP10PK. BEK293 (lanes 2, 3, 6, and 7) and JHLA1 (lanes4, 5, 8, and 9) cells were treated with 250 nM STS (lanes 3 and 4) or300 nM D-Mann (lanes 7 and 8) or mock-treated with DMSO (control forSTS; lanes 2 and 5) or MEM (control for D-Mann; lanes 6 and 9).

[0033]FIG. 3A is an image of an immunoblot depicting inhibition ofcaspase activation by ICP10PK. Proteins obtained from extracts of HEK293(lanes 1 and 2), JHL15 (lanes 3 and 4), and JHLA1 (lanes 5 and 6) cells,mock-treated (lanes 1, 3, and 5) or treated (24 hours) with 250 nM ofSTS (lanes 2, 4, and 6) were resolved by SDS-PAGE (7% acrylamide gels),transferred to nitrocellulose, and immunoblotted with caspase3p32antibody (specific for the inactive caspase-3 pro-enzyme).

[0034]FIG. 3B is a graph depicting inhibition of caspase activation byICP10PK. HEK93, JHL15, and JHLA1 cells treated with STS (as described inFIG. 3A) were stained with caspase3p20 antibody (specific for theactivated caspase-3 which is the large fragment of the pro-enzyme), andcells in five randomly chosen microscopic fields were counted. Resultsare expressed as mean percentage of positive cells ± SEM (*=p<0.01 vs.HEK294; +=p<0.01 vs. JHL15 by Student test).

[0035]FIG. 3C is an image of an immunoblot depicting inhibition ofcaspase activation by ICP10PK as evidenced by an assay which measuresPARP cleavage. The blot in FIG. 4A was stripped and immunoblotted withanti-PARP antibody. The 85 kDa band consistent with the PARP cleavageproduct was seen in STS-treated HEK293 (lane 2) and JHL1 5 (lane 4), butnot in JHLA1 (lane 6) cells.

[0036]FIG. 4A is a schematic representation of the viral mutants,ICP10deltaPK and ICP10deltaRR.

[0037]FIG. 4B is a graph depicting virus replication kinetics inhippocampal neurons. Primary cultures of hippocampal neurons wereinfected with HSV-2, HSV-1, ICP10deltaPK, and ICP10deltaRR at 10PFU/cell. Viral titers were determined at various times between 0 and 48hours post infection by plaque assay. Results are expressed as meanPFU/ml±SEM.

[0038]FIG. 5A is a graph depicting anti-apoptotic activity of ICP10PK invirus-infected hippocampal cultures. Primary hippocampal cultures wereinfected with HSV-2, ICP10deltaPK, ICP10deltaRR, or HSV-1 at 10 PFU/celland analyzed by TUNEL at 24 hours post infection. Apoptotic(TUNEL-positive) and non-apoptotic (TUNEL-negative) cells were countedin five randomly chosen microscopic fields and the results are expressedas mean percentage of apoptotic cells ±SEM. (*=p<0.01 vs. HSV-2;#=p<0.05 vs. HSV-2; +=p>0.05 vs. ICP10deltaPK).

[0039]FIGS. 5B through 5D are images of photomicrographs depictingHoeschst staining of nuclei in cells infected with ICP10deltaPK, HSV-1,and HSV-2, respectively, as described in FIG. 6A.

[0040]FIG. 5E is a graph depicting inhibition of caspase-3 activationfollowing infection with HSV-2, ICP10deltaPK, ICP10deltaRR, or HSV-1.Cultures in FIG. 5A were stained with caspase-3p20 antibody and countedin five randomly chosen microscopic fields. Results are expressed asmean percentage of positive cells ±SEM (*=p<0.05 vs. HSV-2).

[0041]FIGS. 6A trough 6F are a series of images of photomicrographsdepicting neuron-specific antibody staining of TUNEL-positive,virus-infected cells. Primary hippocampal cultures were infected for 24hours at 10 PFU/cell with positivc cells (FITC lIbeled; Figyures 6A and6D)X cells labeled wvitl thie neuron- specific PE-conjulgated TUJ 1(class III beta tubulin) antibody (FIGS. 6B and 6E), and labelco-localization (FIGS. 6C and 6F) are shown,i.

[0042]FIG. 7A is an image of an immunoblot depicting activation of theMEK/MAPK pathway by HSV-2. Primary cultures of hippocampal neurons wereinfected at 10PFU/cell with HSV-2, ICP10deltaPK, or HSV-1 ormock-infected with growth medium and harvested and 0.5 and 24 hours p.i.Proteins were resolved by SDS-PAGE (8.5% acrylamide gels), transferredto nitrocellulose membranes, and immunoblotted with antibody specificfor P-MAPK1/2. Blots were stripped and re-blotted with antibody toMAPK1/2, and protein levels were quantitated by densitometric scanning.

[0043]FIG. 7B is a graph depicting the results of densitometric scanningof bands in FIG. 8A. Results are expressed as ratios of P-MAPK1/MAPK1(MAPK1) and P-MAPK2/MAPK2 (MAPK2).

[0044]FIG. 7C is an image of an immunoblot depicting the ability of theMEK-specific inhibitor, U0126, to inhibit activation of P-MAPK1/2 byHSV-2. Extracts obtained from cells mock-infected (lane 1) or infectedwith HSV-2 in the absence (lane 2) or presence (lane 3) of 20 micromolesof U0126, were immunoblotted with antibody specific for P-MAPK1/2 (upperbands) or MAPK1/2 (bottom bands).

[0045]FIG. 7D is a graph depicting the results of densitometric scanningof bands in FIG. 8C. Results are expressed as ratios of P-MAPK1/MAPK1(MAPK1) and P-MAPK2/MAPK2 (MAPK2).

[0046]FIGS. 7E and 7F are graphs depicting the effect of LY294002 andU0126 inhibitors (which are respectively specific for P13-K/Akt and MEK)on HSV-2-induced apoptosis. Hippocampal cultures were either mockinfected or infected with HSV-2 (10 PFU/cell; 24 hrs) in the absence orpresence of 0-100 micromolar LY294002 or 0-20 micromolar U0126, andanalyzed by TUNEL. Cells were counted in five randomly chosenmicroscopic fields and the results are expressed as mean percentageTUNEL-positive cells +SEM (*=p<0.01 vs. untreated HSV-2 infected cells).antibody neutralized HSi-2 to activate AII. Extracts obtained fi-onicells mocki- infected (lane 1) or infected with FiS/-2 (lane 2) orHSV′-2 neutralized with the IgG flaction fri-om a gD monoclonal antibody(lane 3) were inmllunoblotted with antibody specific for P-MAPK1/2(upper bands) or MAPKl/2 (bottom bands).

[0047]FIG. 5B is a graph depicting the results of densitometric scanningof the bands in FIG. 8A. Results are expressed as ratios ofP-MAPK1/NMAPK1 (MAPK1) and P-MAPK2/MAP2 (MAPK2).

[0048]FIG. 5C is an image of an iminunoblot the inability of HSV-2neutralized with anti-HSV-2 (hyperimmune) seruni to activate MAPK.Extracts obtained from cells mock-infected (lane 1) or infected withHSV-2 neutralized with preimmune (lane 2) or HSV-2 hyperimmune (lane 3)serum were inimunoblotted with antibody specific for P-MAPK1/2 (upperbands) or MAPK (bottom bands).

[0049]FIG. 8D is a graph depicting the results of densitometric scanningof the bands in FIG. 5C. Results are expressed as ratios ofP-MAPK1/MAPK1 (MAPK1) and P-MAPK2/MAPK2 (MAPK2).

[0050]FIG. 9 is an image of an immunoblot depicting expression of ICP10and mutant forms of ICP10 in primary cultures of hippocampal neurons.Extracts from cells mock-infected (lane 1) or infected (10 PFU/cell; 0.5hours) with HSV-2 neutralized with gD monoclonal antibody (lane 2),HSV-2 (lane 3), ICP10deltaPK (lane 4), or ICP10deltaRR (lane 5) wereimmunoblotted with ICP11 antibody. Blots were stripped and reprobed withactin antibody as a control for loading and protein expression.

[0051]FIGS. 10A through 10C are a series of images of photomicrographsdepicting expression of ICP10PK and p139™ in transfected PC12 cells.Differentiated PC12 cells non-transfected, transfected with pJW17 orpJHL15 (FIGS. 10A, 10B, and 10C, respectively) were fixed with 3%paraformaldehyde at 24 hours post-transfection and stained withICP10-specific antibody by immunohistochemistry. Staining is cytoplasmic(arrowhead). Nuclei are counterstained with hematoxylin (arrow).

[0052]FIG. 11 is a graph depicting the ability of ICP10PK to protectPC12 cells from death induced by NGF withdrawal. PC12 cells weredifferentiated by growth (at least twelve days) in serum-free mediumsupplemented with 100ng/ml of NGF. Cells were transfected withexpression vector pJW17 (ICP10PK; solid triangles) or pHL15 (p139TM:open triangles) or not transfected (open squares). Cell viability isexpressed as percentage relative to the number of cells at 0 hours postNGF withdrawal ± SEM (*=p<0.05 vs. control +=p<0.05vs.pJHL15-transfected cells, by ANOV7A witlh Tukey-KIamer post-test).

[0053]FIG. 12 is a graph depicting the ability of ICP I OPK to rescuehippocaipal neurons from death due to growth factor deprivation. Mousehippocampal neurons were plated on glass coverslips, maintained for twodays in MEM with B27 supplement (Gibco BRL, Gaithersburg, Md.) whichcontains optimized concentrations of neuron growth factors, transfectedwith pJW17 (solid triangles) or pJHL15 (open triangles), and the mediumwas replaced with MEM free of serum or B27. Non-transfected cells weremaintained in MEM (Eu-B27; open squares) or MEM with B27 (Eu+B27; solidcircles). Viability was determined by counting live neurons andexpressed as a percentage relative to the initial (t=0) number of viablecells ±SEM (*=p<0.05 vs. control, Eu-B27; +=p<0.05 vs.pJHL15-transfected cells, but ANOVA with Tukey-Kramer post-test).

[0054]FIG. 13 is a graph depicting the ability of ICP10PK to block thedeath of Ts16 hippocampal neurons. Ts16 mouse hippocampal neurons weregrown as described in FIG. 12, and transfected with pJW17 (solidtriangles), pJHL15 (open triangles), or non-transfected (open circles).Non-transfected euploid hippocampal cells maintained in B27-supplementedmedium (Eu+B27; solid circles) were used as a control. Viability wasdetermined by counting live neurons and expressed as a percentagerelative to the initial (t=0) number of viable cells ±SEM (*=p<0.001 vs.control Ts16 non-transfected; +=p<0.001 vs. pJHL15-transfected cells, byANOVA with Tukey-Kramer post-test).

[0055]FIGS. 14A through 14C is a series of images of photomicrographsdepicting the morphology of Ts16 hippocampal neurons. Ts16 mousehippocampal neurons were grown and transfected with pJW17, pJHL15, orremained non-transfected (FIGS. 14A, 14B, and 14C, respectively).Micrographs were taken by using phase-contrast microscopy at 72 hourspost-transfection (five days in vitro).

[0056]FIG. 15 is a graph depicting the ability of ICP10PK to inhibitapoptosis in Ts16 hippocampal neurons. Ts16 mouse hippocampal neuronswere grown and transfected with pJW17, pJHL15, or remainednon-transfected. Non-transfected euploid hippocampal cells maintained inB27-supplemented medium 0 I(12/11777 (i 1(I II/23545 (Eu+13277) vCIcised ;s a contiol.].euslts of TU-TT?L it 72 IIouL-s post-tl-alisfoctioll(five days in vitro) are expressed as perceniage of ipoptotic(TWiT.EL-positive) cells +SENI (=p<O.Ol vs. control Tst6non-trainsfected; -1-=p<0.01 vs. pJIILI5-transfected cells, by ANOVAwith Tukey-Kral-ler post-test).

[0057]FIG. 16 an image of a photomicrograph depicting staining ofICP10PK in mouse hippocampal neurons. Mice (C57/B1) were infectedintranasally with ICP10deltaRR or phosphate buffer saline (PBS;control). At seven days post-infection, mice were anesthetized withether and perfused with 4% paraformaldehyde. Coronal brain sections (20micrometer) were immunostained with antibody specific for ICP10 by theimmunoperoxidase method. Staining was observed in the hippocampi of miceinfected with ICP10deltaRR virus, but not in mock (PBS)-infected mice.

DETAILED DESCRIPTION OF THE INVENTION

[0058] The invention relates to the discovery of the anti-apoptoticactivity of HSV-2 ICP10PK and its use as an inhibitor of apoptosis ofneuronal (as well as non-neuronal) cells in a mammal, including mammalssuffering from neurodegenerative disorders and acute diseases involvingneuronal degeneration. The invention discloses a neuroprotective rolefor ICP10, based on its ability to protect neurons from apoptosisinduced by a variety of stimuli.

[0059] As disclosed herein, the PK domain of ICP10 (ICP10PK) inhibitsapoptosis, as characterized by a reduction in the number of TdT-mediateddUTP nick end labeled (TUNEL)-positive cells, inhibition of DNAfragmentation, and inhibition of cellular morphologies characteristic ofapoptosis. The anti-apoptotic activity of ICP10PK is shown herein to beeffective against caspase-3-dependent apoptosis and is shown to involveactivation of the MEK/MAPK pathway. ICP10PK blocks apoptosis induced byvarious chemical apoptotic inducers, including staurosporine (STS) andD-mannitol (D-Mann), as well as growth factor withdrawal, virus-inducedapoptosis and apoptosis resulting from genetic defects.

[0060] Useful in the invention is an isolated nucleic acid encodingHSV-2 ICP10 (SEQ ID NO:1). The HSV-2 ICP10 gene (GeneBank No. M12700) isdescribed in U.S. Pat. Nos. 6,013,265, 6,054,131, and 6,207,168, each ofwhich is incorporated herein in their entirety.

[0061] In some aspects of the invention, mutants variants, homologs, orfragments of the isolated nucleic acid encoding ICP10 are useful. Asdisclosed herein, fragments of the isolated nucleic acid which encodeonly the PK domain of ICP10 arc useful in the invention. 1 nl oneexample, the ribonucleotide reductase (RR! domain is deleted from ICP10.Elimination of this domain is useful in that the resulting mutant isrendered non-virulent while it retains anti-apoptotic activity and theviral RR will not interfere with cellular processes governed by the RRencoded by the cell. Fragments of the nucleic acid encoding ICP10 can begenerated by standard molecular biology procedures known in the art, forexample in Sambrook et al. (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), in Ausubel et al.(1997, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork), and in Gerhardt et al. (eds., 1994, Methods for General andMolecular Bacteriology, American Society for Microbiology, Washington,D.C.). Further, the anti-apoptotic activity of ICP10 can be tested incultured cells using the methods described in the examples herein.

[0062] Assays that measure induction of apoptosis in cells in vitro andex vivo are described and used herein. These assays include TUNEL, DNAfragmentation assay, cell viability counts, HOECHST staining, andWestern blots to measure inhibition of caspase activation and PARPcleavage.

[0063] Also useful in the invention is a vector comprising the nucleicacid encoding ICP10. Given the neurotropism of HSV-2, this virus servesas a useful vector for delivery of ICP10 to neurons. Particularly usefulin the invention, is an ICP10-encoding HSV-2 vector wherein the RRdomain of ICP10 has been deleted (ICP10deltaRR), thereby rendering thevirus replication-defective but retaining the anti-apoptotic activity ofthe PK domain of ICP10. Other viral and non-viral vectors encoding ICP10may also be useful in the invention. For example, retrovirus vectorsexpressing ICP10 or ICP10PK can be used to stably infect neuronal stemcells useful in ex vivo gene therapy. Other viral vectors including, butnot limited to, adenovirus, vaccinia virus, canary pox virus, and adenoassociated virus are useful for delivery and expression of ICP10 orICP10PK using in vivo gene therapy.

[0064] Vectors encoding ICP10 can be constructed by standard molecularbiology techniques. An HSV-2 vector, ICP10deltaRR, wherein the RR domainof ICP10 was replaced with a nucleic acid encoding LacZ was constructedpreviously (U.S. Pat. Nos. 5,965,356, 6,013,265, 6,054,131, and6,207,168). Construction of ICP10deliaRR is described further in theexamples presented herein. Other HSV-2 vectors encoding mutants,variants, homologs, or fragments of ICP10 can be constructed by similarmethods.

[0065] Also useful in the invention is an ICP10 nucleic acid sequenceoperably linked to a promoter regiulator) sequence that facilitatesexpression of ICP10. It may be particularly useful to link the nucleicacid encoding ICP10 to a tissue specific or an inducible promoter.Because the invention relates to the expression of ICP10 in neuronalcells, neuron-specific promoters will be useful to induce the expressionof ICP10 specifically in these cells. Promoters useful to the inventionare specific for neuronal cells; these include the neuron-specificenolase (NSE) and tyrosine hydroxylase (TH) promoters, TH-NFH(neurofilament heavy subunit) chimeric promoter, and the golli promoter;each of these promoters is described in detail below. Endogenousmammalian NSE is expressed in essentially all neurons, beginning duringdevelopment at the time of synaptogenesis; its activity increases at asteady rate into adulthood when amounts of this protein can reach levelsof up to 1% of the total cell protein (Marangos et al., 1987, Ann. Rev.Neurosci., 60:269-295). The pattern of expression of this promoter makesit a good candidate for conferring long-term expression of foreign geneson adult neurons following delivery by a viral vector. The TH-NFHpromoter supports long-term gene expression in striatal neurons (Wang etal., 2001, Biotechniques, 31:204-212). Golli products of the myelinbasic protein (MBP) gene have been found to be expressed in neuronsduring postnatal and embryonic development including Cajal-Retzius andcortical subplate neurons. Moreover, golli expression occurrs in othercortical neurons including neurons from cortical layer V and thehippocampus (Pribyl et al., 1996, J. Comp. Neurol., 374:342-353; Pribylet al., 1993, Proc. Natl. Acad. Sci. USA, 90:10695-10699). Consequently,the golli promoter may be useful for driving transgene expression inselected neuronal populations.

[0066] Viral promoters including the HSV latency associated transcript(LAT) promoter, the Moloney murine leukemia virus (Mo-MLV) long terminalrepeat (LTR), and the human cytomegalovirus (HCMV) immediate early (IE)promoter may also by useful. The LAT promoter includes elements bothupstream and downstream of the start site of the minor LAT mRNA fromwhich the intranuclear LATs are derived. Promoter elements referred toas LAP2 (latency active promoter 2) and LAP1 (contains neuronalresponsive elements) are independently capable of expressing LAT duringviral latency in sensory ganglia. The transgene can be placed downstreamof LAPS near the start of the LAT mi A or downstream of both promoterswvitllin the LAT intron. Stable transgene expression has been achievedin sensory ganglia, but expression in CNS neurons was less vigorous(Fink et al., 1997, Nature Med, 3:357-359). The LTR of Mo-MLV has beenused with HSV vectors to yield stable expression of the LacZ gene insensory neurons and extended expression in motor neurons of thehypoglossal nucleus (Dobson et al., 1990, Neuron, 5:353-360). The HCMVIE promoter is a very strong constitutive promoter that is active in awide variety of cell types including CNS neurons both in vitro (Johnsonet al., 1992, Mol. Brain Res., 12:95-102) and in vivo (Wood et al.,1994, Exp. Neurol., 130:127-140). The vectors described above may alsocomprise such promoters operably linked to the ICP10 nucleic acid.

[0067] Also useful to the invention is an isolated ICP10 polypeptide ora mutant, variant, homolog, or fragment thereof having theanti-apoptotic activity of ICP10, as described herein. As describedabove, fragments of ICP10 in which the RR domain has been deleted areshown herein to be useful in the invention.

[0068] ICP10 polypeptides or mutant, homolog, or fragments thereof canbe delivered using standard methods of peptide delivery known in theart. Specifically, the use of liposomes may be useful for ICP10 peptidedelivery.

[0069] Also useful in the invention is an isolated nucleic acid encodingbcl-2 (GenBank No. M14745) in combination with an isolated nucleic acidencoding ICP10. Similar to ICP10, bcl-2 has anti-apoptotic activity.However, ICP10 and bol-2 inhibit apoptosis via distinct pathways.ICP10PK acts as a growth factor receptor, signalling at the cellularmembrane via Raf, MEK, and ERK, and bcl-2 appears to function muchfurther downstream in an apoptotic inhibition pathway. In addition, themechanism of apoptosis inhibition appears to be different. WhereasICP10PK blocks cytochrome c release from the mitochondria by activatinga protein other than bcl-2 (BAG-1) which ultimately leads to inhibitionof caspase activation. In contrast, bcl-2 appears to function fardownstream (directly at the level of the mitochondria) to inhibitcytochrome c release. Therefore, ICP10PK functions to activatedownstream targets other than bcl-2 and has a significantly broaderapoptotic activity than bcl-2. Since these proteins function throughapparently distinct pathways, the use of both to inhibit apoptosis islikely to have a synergistic effect.

[0070] A number of ICP10 and bel-2 nucleic acid combinations are usefulin the invention. For example an isolated utit(;Ilct tcid enrcodingICP10 miiy be delivered to a neuron in combination Ntit an isolatednucleic acid encoding bcl-2. Alternatively, the two nucleic acids may belinked using standard molecular biology techniques and delivered as asingle fused nucleic acid molecule. Further, fragments of eithermolecule may be delivered, wherein each fragment retains biologicalactivity of the respective protein encoded thereby.

[0071] The biological activity of TCP10PK differs from that of bcl-2 inthat, although both inhibit apoptosis. ICP10PK functions at thebeginning of the apoptotic signaling cascade and results in theactivation of end points other than bcl-2, such as bag-1, and bcl-2functions at the end of the cascade, inhibiting cytochrome c releasefrom the mitochondria. Thus, the biological activity of ICP10PK isdefined as the ability to inhibit apoptosis in a cell at the beginningof the apoptotic signaling cascade. In contrast, the biological activityof bcl-2 is defined as the ability to inhibit apoptosis by directlyblocking cytochrome c release from the mitochondria.

[0072] The bcl-2 family of proteins acts at the mitochondrial level toprevent mitochondrial dysfunctions such as membrane potential loss andthe membrane permeability transition, which allows the release of themitochondrial apoptosis-inducing factor (AIF) and cytochrome c, aprocess that initiates the apoptotic cascade. Moreover, bcl-2 familymembers sequester the proforms of caspases, inhibiting their activation.Conversely, bcl-2 has a broad anti-apoptotic activity (Tsujimoto, 1998,In: Apoptosis: Mechanisms and Role in Disease, 137-155). There are,however, pathways to apoptosis that are insensitive to bcl-2, such asapoptosis induced by activation of CD95 and TNF receptors (Strasser etal., 1995, EMBO J., 14:6136-6147). Moreover, bcl-2 is overexpressed inAD brains (Kitamura et al., 1998, Brain Res., 780:260-269), a processwhich cannot, however, circumvent neuronal death. Bcl-2 does not protectagainst motoneuron degeneration in wobbler and progressive motorneuropathy (Sagot et al., 1995, J. Neurosci., 15.:7727-7733), whichsuggests that bcl-2 alone would not be sufficient for treatment ofneurodegenerative disorders.

[0073] By contrast, ICP10PK activates cellular signaling pathways suchas Ras/MEK/MAPK which are involved in survival as well as in maintaininga normal neuronal physiology. As such, Ras/MEK/MAPK promotes survival bytranscription-independent (phosphorylation of pro-apoptotic protein Badby Raf which changes its status from pro- to anti-apoptotic) ortranscription-dependent (increased transcription '000, CiiT. Opin.Nieurobiol., 10:3S1-391). Besides direct activation of the cell proteinsynthesis macl-ineiy, Mrss are also involked in modulation ofion-cliannel function (Grewal et al., 1999, CuiT. Opin. Neurobiol.,9:544-553) and plhosplhorylation of synapsin I (Jovanovic et al., 1996,Proc. Natl. Acad. Sci. USA, 93:3679-3683) and these functions mayrepresent mechanisms by which neurotrophins exert rapid effects onneurotransmission. MAPKs are required for hippocampal long termpotentiation (LTP) (English et al., 1997, J. Biol. Chem.,272:19103-19106) which represents the molecular mechanism that underlieslearning and memory. Compelling evidence for a MAPK-dependent role inlearning and memory has come from rodent behavioral studies, wherebehavioral performance was associated with increased MAPK activity, andinhibition of MAPK signaling specifically impaired learning (Blum etal., 1999, J. Neurosci., 19:3535-3544; Berman et al., 1998, J.Neurosci., 18:10037-10044; Atkins et al., 1998, Nat. Neurosci.,1:602-609). Taken together, these data provide strong evidence that theRas/MEK/MAPK pathway, in addition to conducting survival signals, mayalso be required for both synaptic plasticity and learning and memory.

[0074] Also useful in the invention are vectors comprising nucleic acidsencoding ICP10 and bcl-2. The nucleic acids may be present withinseparate vectors or within the same vector. When the nucleic acids arewithin the same vector, the nucleic acids may be polycistronic such thattheir expression is linked to one another or they may be expressedindependently from one another. Many vectors may be useful fordelivering the combination of ICP10 and bcl-2 to cells in a mammal. In apreferred aspect of the invention, a recombinant HSV-2 vector in whichthe RR domain of ICP10 has been replaced with the nucleic acid encodingbcl-2 is used to deliver ICP10 and bcl-2 to neuronal cells. One versionof this virus will express an ICP10-bcl-2 fusion protein, whereasanother version will express the two proteins separately. Constructionof these viruses is described in the examples section herein.

[0075] In the methods of the invention, ICP10 may be delivered toneuronal cells in the form of a nucleic acid expressing ICP10,preferably using vectors or liposomes, or it may be delivered to cellsin the form of a polypeptide, or a fragment thereof. Thus, the use ofICP10 polypeptide (SEQ ID NO:2) and fragments thereof, including allmutants and variants having IPC10 biological activity as defined here,are included in the methods of the invention. ICP10 polypeptides can beeasily generated using methods well known in the art described, forexample, in Sambrook U.S. Pat. Nos. 6,013,265, 6,054.131, and 6,207,168

[0076] Also useful in the methods of the invention is the use of acombination or CP10 and bcl-2 polypeptides. The two proteins can bedelivered independently or together as a fusion protein. An ICP10-bcl-2fusion protein can be constricted using standard techniques known in theart wherein the nucleic acids encoding each protein are operably linkedas described above. The combination of the ICP10 and bcl-2 polypeptidesor fusion polypeptides may be delivered to neuronal cells by the methodsdescribed above for delivery of ICP10 protein alone.

[0077] ICP10 is shown herein to protect hippocampal neurons from deathinduced by withdrawal of neurotrophic support. Furthermore, ICP10rescued mouse Ts16 cells, a naturally occurring model for DS, fromapoptosis, demonstrating the anti-apoptotic activity of ICP10specifically with respect to genetic defects associated withneurodegeneration. Given that many clinically-significantneurodegenerative disorders are characterized by neuronal apoptosis, theinvention makes use of the anti-apoptotic activity of ICP10 to treatsuch disorders, including, but not limited to, AD, ALS, DS, PD, and HD.Notably, the therapeutic value of ICP10 for treatment ofneurodegenerative disorders is further substantiated by thedemonstration herein that peripheral administration using a viral vectoreffectively delivers ICP10 to the central nervous system (CNS). Thus,the data presented herein demonstrate the usefulness of ICP10 ininhibiting neuronal apoptosis, including that associated withneurodegenerative disorders.

[0078] The invention includes a method of inhibiting apoptosis ofneuronal cells in a mammal. The method comprises administering to themammal an apoptosis-inhibiting amount of an isolated nucleic acidencoding ICP10. The invention should be construed to includeadministration of alternative forms of ICP10 having anti-apoptoticactivity, including mutants, variants, homologs, and fragments of ICP10.Specifically, fragments of ICP10 which retain the PK domain of ICP10 andthus retain the anti-apoptotic activity of ICP10 are useful forinhibiting cellular apoptosis, as disclosed herein. The anti-apoptoticactivity of ICP10 maps to the PK domain (amino acids 1 to 411), andthus, fragments useful to the invention will comprise this domain.

[0079] The isolated nucleic acid encoding ICP10 can be administered to amammal using a variety of methods. In a preferred embodiment of theinvention, ICP10 is delivered using a vector. Numerous vectors are knownin the art including, ic:PI( ) is delivered Lsing a vector. Nulimerousvectoi-s zirc kinoxyni in the l-lI incllidiln”, but not limited to,linear-1)olyntiucleotides, polrnucleotides associated vitli ionic oramphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term shouldalso be construed to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example,polylysine compounds, liposomes, and the like. Examples of viral vectorsinclude, but are not limited to, herpesvirus vectors, adenoviralvectors, adeno-associated virus vectors, retroviral vectors, and thelike.

[0080] The present invention also provides for a method of inhibitingapoptosis using analogs of proteins or peptides encoded by ICP10.Analogs can differ from naturally occurring proteins or peptides byconservative amino acid sequence differences or by modifications whichdo not affect sequence, or by both.

[0081] For example, conservative amino acid changes may be made, whichalthough they alter the primary sequence of the protein or peptide, donot normally alter its function. Conservative amino acid substitutionstypically include substitutions within the following groups:

[0082] glycine, alanine;

[0083] valine, isoleucine, leucine;

[0084] aspartic acid, glutamic acid;

[0085] asparagine, glutamine;

[0086] serine, threonine;

[0087] lysine, arginine;

[0088] phenylalanine, tyrosine.

[0089] Modifications (which do not normally alter primary sequence)include in vivo, or in vitro chemical derivatization of polypeptides,e.g., acetylation, or carboxylation. The invention should be construedto include administration of modified ICP10 peptides including, but notlimited to, peptides modified by glycosylation, e.g., those made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing or in further processing steps; e.g., byexposing the polypeptide to enzymes which affect glycosylation, e.g.,mammalian glycosylating or deglycosylating enzymes. Also embraced is amethod of inhibiting apoptosis comprising administration of ICP10peptides which have phosphorylated amino acid residues, e.g.,phosphotyrosine, phosphoserine, or phosphothreonine.

[0090] The invention further includes a mthod of inhibiting apoprosis byadministering ICP10 polypeptides which have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as a therapeutic agent. Analogs of suchpolypeptides include those containing residues other than naturallyoccurring L-amino acids, e.g., D-amino acids or non-naturally occurringsynthetic amino acids. The peptides of the invention are not limited toproducts of any of the specific exemplary processes listed herein.

[0091] Pharmaceutical compositions comprising ICP10 nucleic acid,vectors comprising the same, or peptides encoded thereby, may beformulated and administered to a mammal for inhibition of apoptosis.Such compositions are now described.

[0092] The invention encompasses the preparation and use ofpharmaceutical compositions comprising an ICP10 compound useful forinhibition of apoptosis as an active ingredient. Such a pharmaceuticalcomposition may consist of the active ingredient alone, in a formsuitable for administration to a subject, or the pharmaceuticalcomposition may comprise the active ingredient and one or morepharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

[0093] As used herein, the term “pharmaceutically acceptable carrier”means a chemical composition with which the active ingredient may becombined and which, following the combination, can be used to administerthe active ingredient to a subject.

[0094] As used herein, the term “physiologically acceptable” ester orsalt means an ester or salt form of the active ingredient which iscompatible with any other ingredients of the pharmaceutical composition,which is not deleterious to the subject to which the composition is tobe administered.

[0095] The formulations of the pharmaceutical compositions describedherein may be prepared by any method known or hereafter developed in theart of pharmacology. In general, such preparatory methods include thestep of bringing the active ingredient into association with a carrieror one or more other accessory ingredients, and then, if necessary ordesirable, shaping or packaging the product into a desirable single- ormulti-dose unit.

[0096] Although the descriptions of pharmaceutical compositions providedherein are principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and other mammals.

[0097] Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor parenteral, topical, pulmonary, intranasal, buccal, ophthalmic,intrathecal or another route of administration.

[0098] A pharmaceutical composition of the invention may be prepared,packaged, or sold in bulk, as a single unit dose, or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

[0099] As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

[0100] Formulations of a pharmaceutical composition suitable forparenteral administration conprise the active ingqredient combined witlha pharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Suclh fonmulations may be prepared, packaged, or soldin a form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

[0101] Pharmaceutical compositions of the invention formulated forpulmonary delivery may also provide the active ingredient in the form ofdroplets of a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers. Theformulations described herein as being useful for pulmonary delivery arealso useful for intranasal delivery of a pharmaceutical composition ofthe invention.

[0102] Typically dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1microgram to about 100 grams for proteins and peptides, 10³ to 10⁸plaque forming units for viruses, and 1 to 500 micrograms for DNA.

[0103] The compound may be administered to an animal as frequently asseveral times daily, or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenless frequently, such as once every several months or even once a yearor less. The frequency of thedose will be readily apparent to theskilled artisan and will depend upon any number of factors, such as, butnot limited to, the type and severity of the disease being treated, thetype and age of the animal, etc.

[0104] For example, treatment of AD, a chronic disease, may be performedas follows. ICP10deltaRR can be given by intranasal spraying, anon-invasive and widely accepted delivery route, although other routesof administration are possible. As stated above, 10³ to 10⁸ plaqueforming units of ICP10deltaRR can be used for infection. Assuming thatgene expression does not last more than 20 days, monthly re-exposurewill be needed (or at least 10 exposures per year).

[0105] To treat an acute disease, such as stroke, ICP10delta RR can beadministered as described above. Again assuming that gene expressiondoes not last more than 20 days, re-exposure will only be needed 2 or 3additional times (4 exposures total).

[0106] Also included in the invention is a method of inhibitingapoptosis of neuronal cells in a mammal by administering ICP10 incombination with bcl-2. Like ICP10, bcl-2 inhibits apoptosis; however,the mechanism of inhibition appears to be distinct from that ofICP10.Thus, administration of ICP10 and bcl-2 will have a cumulativeanti-apoptotic activity due to the ability of these two proteins toactivate distinct survival pathways. The invention should be construedto include administration of ICP10 and bcl-2 nucleic acids, a vectorcomprising ICP10 and bcl-2 nucleic acids, and the ICP10 and bcl-2polypeptides encoded thereby as well as pharmaceutical compositionscomprising the same, as described herein. Fusion proteins comprisingportions of ICP10 and bcl-2 are also considered in the invention,wherein the administration of the fusion protein or a nucleic acidencoding the fusion protein inhibits apoptosis in a tissue in a mammal.In additional embodiments, members of the bcl-2 family withanti-apoptotic activity that are not activated by ICP10PK may be usefulfor combination delivery. Specifically, these can be delivered aspeptide cocktails, mixed DNA preparations, or other fusion proteins withICP10 expressed by the ICP10deltaRR mutant virus, as two such genescould likely be incorporated into the virus.

[0107] In a preferred embodiment of the invention, ICP10 is used toinhibit neuronal apoptosis in a mammal suffering from aneurodegenerative disorder. Neurodegenerative diseases amenable totreatment with ICP10 include AD, ALS, DS, PD, and HD. Some examples ofdiseases which may be treated according to the methods of the inventionare discussed herein. The invention should not be construed as beinglimited solely to these examples, as other neurocdegeinerative diseaseswich are at present cueLlaov,l, once knlown, may also be treatable usingthe methods of the invention.

[0108] Examples of acute diseases that could be treated with ICP10PKinclude stroke, cerebral ischemia, chemical and/or physical trauma, andspinal cord injury. Patients suffering any of these injuries experienceneuronal apoptosis and may be treated effectively with ICP10PK. Thesetypes of injuries require treatment within days, if not hours of the.injury and are excellent candidates for the anti-apoptotic use ofICP10PK. Thus, administration of ICP10PK is useful in inhibitingapoptosis in both the central nervous system as well as the peripheralnervous system, where it will be particularly effective in cases ofspinal cord injury and diabetic neuropathy.

[0109] Preferred methods of ICP10 administration specifically for thepurpose of inhibiting neuronal apoptosis in a mammal include intranasaladministration and subcranial injection of a nucleic acid encodingICP10, a vector comprising the same, or a polypeptide encoded thereby.The use of liposomes to deliver ICP10-encoding nucleic acids may also beuseful in the invention.

[0110] In a preferred embodiment of the invention, ICP10 is administeredusing a mutant form of HSV-2, designated ICP10deltaRR, in which theribonucleotide reductase domain of ICP10 is replaced with the geneencoding LacZ. The ICP10deltaRR virus is known in the art (Peng et al.,1996, Virology, 216:184-196; U.S. Pat. Nos. 5,965,356, 6,013,265,6,045,131, and 6,207,168). LacZ-specific staining facilitates detectionof cells that are infected with the mutant virus, and more specifically,cells that are expressing ICP10. ICP10deltaRR can also have a deletionof the RR gene, without the addition of the LacZ gene. One could replacethe LacZ gene with bcl-2 or any other gene of interest, for example, agrowth factor.

[0111] The invention further includes a method of administering ICP10 incombination with bcl-2 to inhibit apoptosis of neurons in a mammal. Asdescribed in detail above, since ICP10 and bcl-2 inhibit apoptosisthrough distinct pathways, the combination of ICP10 and bcl-2 will havea cumulative effect. Inhibition of neuronal apoptosis may be achieved byadministration of ICP10 and bcl-2 nucleic acids, a vector comprisingICP10 and bcl-2 nucleic acids, and the ICP10 and bcl-2 polypeptidesencoded thereby, as well as pharmaceutical compositions comprising thesame as described herein. Again, fusion proteins comprising portions ofICP10 and bcl-2 ale also considered in the invention, wherein theadministration of the fusion protein or a nucleic acid encoding thefusion protein inhibits neuronal apoptosis in a mammal.

[0112] In a preferred embodiment, ICP10 and bcl-2 are administered incombination to inhibit neuronal apoptosis using an HSV-2 mutant virus inwhich the ribonucleotide reductase domain of ICP10 has been replacedwith the bcl-2 gene. A mutant virus in which the RR domain of ICP10 isreplaced with bcl-2 can be constructed as described in the examplespresented herein.

[0113] Administration of ICP10, or ICP10 in combination with bcl-2 mayalso be useful for treatment of apoptosis in cells other than neurons.As disclosed herein, ICP10PK inhibits apoptosis in human kidney cells(JHLA1 cells). Thus, the invention may also be used to treatnon-neuronal diseases which rely upon inhibition of apoptosis for theirtreatment.

[0114] Definitions

[0115] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element. “Plurality” means at least two.

[0116] As used herein, cells or tissue are “affected” by a disease ordisorder if the cells or tissue have an altered phenotype relative tothe same cells or tissue in a subject not afflicted with a disease ordisorder.

[0117] As used herein, amino acids are represented by the full namethereof, by the three letter code corresponding thereto, or by theone-letter code corresponding thereto, as indicated in the followingtable: Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp DGlutamic Acid Glu E Lysine Lys K Arginine Arg R Histidine His H TyrosineTyr Y Cysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser SThreonine Thr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu LIsoleucine Ile I Methionine Met M Proline Pro P Phenylalanine Phe FTryptophan Trp W

[0118] As used herein, the term “apoptosis” means a process by which acell is affected in such a way that it begins the process of programmedcell death, which is characterized by the fragmentation of the cell intomembrane-bound particles that are subsequently eliminated by the processof phagocytosis. Thus, “inhibition of apoptosis” means reducing oreliminating the apoptotic process in cells.

[0119] “Inappropriate apoptosis” of cells refers to apoptosis (i.e.programmed cell death) which occurs in cells of an animal at a ratedifferent from the range of normal rates of apoptosis in cells of thesame type in an animal of the same type which is not afflicted with adisease or disorder.

[0120] A “coding region” of a gene consists of the nucleotide residuesof the coding strand of the gene and the nucleotides of the non-codingstrand of the gene which are homologous with or complementary to,respectively, the coding region of an mRNA molecule which is produced bytranscription of the gene.

[0121] A “disease” is a state of health of an animal wherein the animalcannot maintain homeostasis, and wherein if the disease is notameliorated then the animal's health continues to deteriorate. Incontrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

[0122] A disease or disorder is “alleviated” if the severity of asymptom of the disease or disorder, the frequency with which such asymptom is experienced by a patient, or both, are reduced.

[0123] “Encoding” refers to the inherent property of specific sequencesof nucleotides in a polynucleotide, such as a gene, a cDNA, or mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

[0124] Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

[0125] As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property by whichit is characterized. A functional enzyme, for example, is one whichexhibits the characteristic catalytic activity by which the enzyme ischaracterized.

[0126] “Homologous” as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., between two nucleicacid molecules, e.g., two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology.

[0127] As used herein, “homology” is used synonymously with “identity”.

[0128] Percent identity of one polynucleotide or polypeptide withrespect to another polynucleotide or polypeptide may be determined usingany available al(gorithlmi, such as the BLAST program.

[0129] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g. gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid or nucleotide residue as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., percent identityis equal to the number of identical positions divided by the totalnumber of positions (e.g., overlapping positions) multiplied by 100). Inone embodiment the two sequences are the same length, at least afterintroducing gaps into one or both sequences.

[0130] Determination of percent identity between two sequences can beaccomplished using a number of mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm used for comparison oftwo sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, gapped BLAST analysis can be used asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. Id. When using BLAST,gapped BLAST, and PSI-Blast analyses, default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm used for comparison of sequences is thealgorithm of Myers and Miller. (19SS) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0) which is part ofthe GCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Yet another useful algorithm for identifying regions of local sequencesimilarity and alignment is the FASTA algorithm as described in Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-244S. When usingthe FASTA algorithm for comparing nucleotide or amino acid sequences, aPAM120 weight residue table can, for example, be used with a k-tuplevalue of 2.

[0131] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, only exact matches arecounted.

[0132] An “isolated nucleic acid” refers to a nucleic acid segment orfragment which has been separated from sequences which flank it in anaturally occurring state, e.g., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, e.g.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g, asa cDNA or a genomic or EDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

[0133] In the context of the present invention, the followingabbreviations for the commonly occurring nucleic acid bases are used.“A” refers to adenosine, “C” refers to cytosine, “G” refers toguanosine, “T” refers to thymidine, and “U” refers to uridine.

[0134] By describing two polynucleotides as “operably linked” is meantthat a single-stranded or double-stranded nucleic acid moiety comprisesthe two polynucleotide arranged within the nucleic acid moiety in such amanner that at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. ByIvay of example, a promoter operably linked to the coding region of agene is able to promote transcription of the coding region.

[0135] The tenm ““nLcleic acid” typically refers to largepolymicleotides.

[0136] The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

[0137] Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

[0138] The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

[0139] “Polypeptide” refers to a polymer composed of amino acidresidues, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

[0140] The term “protein” typically refers to large polypeptides.

[0141] The term “peptide” typically refers to short polypeptides.

[0142] Conventional notation is used herein to portray polypeptidesequences: the left-hand end of a polypeptide sequence is theamino-terminus; the right-hand end of a polypeptide sequence is thecarboxyl-terminus.

[0143] As used herein, the term “promoter/regulatory sequence” means anucleic acid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific mamier.

[0144] An “inducible” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

[0145] A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

[0146] A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell.

[0147] The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

EXAMPLE 1 HSV-2 ICP10PK Inhibits Auoptosis

[0148] The data herein demonstrate a neuroprotective role for ICP10PKexpressed by a viral or expression vector. ICP10PK protects cells fromapoptosis induced by virus, growth factor or genetic defects. Also, itis demonstrated that ICP10PK activates the Ras/MEK/MAPK pathway.

[0149] The materials and methods used in the experiments presented inthis example are now described.

[0150] Cells

[0151] Vero (African green monkey kidney cells) were obtained from ATCCand grown in Minimum Essential Medium (MEM) with 10% fetal bovine serum(FBS) and antibiotics. HEK293 (human embryonic kidney) cells were grownin Dulbecco's Modified Eagle's Medium (DMEM) with 10% FBS, 1 mM sodiumpyruvate, 0.1 mM non-essential amino acids, and antibiotics. The cellline JHLA1 was established from HEK293 cells by transfection with anICP10 expression vector and clone selection with G418, and the cell lineJHL15 was similarly constructed using a vector which expresses thetransmembrane deleted mutant of ICP10, p139 ™, which is PK negative, asdescribed (Smith et al. 1994, Virology, 200:59S-612).

[0152] Primary hippocampal cultures were produced from E17-E18 ratsusing a procedure previously described (Arcava,et al. 1987, FEBS Lett.,229:63-70). Briefly, minced fetal hippocampi were incubated with 0.25%trypsin for 30 minutes at 36° C. followed by dissociation of neurons inMEM supplemented with 10% FBS, 10% horse serum, 2 mM glutamine, and 20microgram/ml DNase II, using a sterile Pasteur pipette. Neurons wereplated at approximately 750,000 cells per 35 mm dish on glass coverslipspre-coated with poly-L-lysine and incubated at 36° C., 10% CO₂. At 24hours, media was replaced with MEM supplemented with 10% horse serum and2 mM glutamine, and the cultures were returned to a 36 degrees C., 10%CO₂ incubator. The results presented in this example were obtained usingneurons at day 6 after plating.

[0153] Rat pheochromocytoma (PC12) cells were obtained from ATCC andwere cultured in DMEM-F12 (Gibco-BRL) with 10% FBS, 0.36% D-glucose(Sigmna), 0.21% sodium bicarbonate, 0.009% gentamycin and 100 ng/mlnerve growth factor (NGF) (Roche Molecular Biochemicals). Generation oftrisomic mice and karyotyping were previously described (Bambrick etal., 1999, J. Neurochem, 72:1769-1772). Primary hippocampal culturesfrom embryonic day 16 (E16) euploid and Ts16 mouse fetuses wereestablished and grown as previously described (Bambrick et al., 1999, J.Neurochem, 72:1769-1772) on glass coverslips etched with a grid of175×175 micrometers squares (CELLocate; Eppendorf, Madison, Wis.) in MEMwith B27 supplement (Gibco) which contains optimized concentrations ofneuron survival factors. For each Ts 16 fetus, an euploid fetus from thesame litter was used.

[0154] The Ts16 mouse is considered to be a model of Down's syndrome(DS; trisomy 21). Ts16, as well as Ts21, are genetic defects believed toconfer increased vulnerability to neurodegeneration (Coyle et al., 1988,Trends Neurosci., 11:390-394). Cultured hippocampal neurons from theTs16 mouse exhibit increased cell death relative to littermate euploidcells, even in the presence of adequate trophic support (Bambrick etal., 1999, J. Neurochem., 72:1769-1772). To examine whether ICP10PK canpromote survival in this system, primary hippocampal cultures from Ts16mice (established as described in Bambrick et al., 1999, J. Neurochem.72:1769-1772) were transfected with pJW17 or pJHL15 at 2 days in cultureand maintained in B27-supplemented medium for the duration of theexperiment.

[0155] Viruses

[0156] HS7-2 (strain G) and HSV-1 (strain F) were used throughout thesestudies (Ejercito et al., 1968, J. Gen. Virol, 2:357-364).

[0157] The HSV-2 ICP10deltaRR mutant was obtained using HSV-2 (G) byreplacing the RR domain of ICP10 with the lacZ gene. The plasmid usedfor insertion of lacZ was previously described (Luo et al., 1992, J.Biol. Chem., 267:9645-9653). The construction of the ICP10deltaRR mutantvirus was reported elsewhere (Peng et al., 1996, Virology, 216:184-196;Smith et al., 1998, J. Virol., 72:9131-9141). Briefly, BamHIE and Tfragments from TP101 plasmid were replaced with a 1.8 kb SaII/BgIIIfragment from plasmid pJHLA9 (Peng et al., 1996, Virology, 216:184-196),containing ICP10 with a deletion in the PK domain. A 10 kb HindIII/EcoRIfragment from the resulting plasmid (containing ICP10deltaPK sequenceflanked by 4 and 2.8 kb HSV-2 DNA sequences at the 5′ and 3′ ends,respectively) was introduced into ICP10deltaRR. The recombinant virussuch as obtained was selected by staining with X-gal (BoehringerMannheim). ICP10deltaPK formed white plaques on a background of blueplaques. White plaques were picked and ICP10deltaPK virus stock wasobtained by growing the virus in Vero cells with MEM, 10% FBS. Virustiters were determined by plaque assay as described (Aurelian, 1992, In:Clinical Virology Manual, 2^(nd) ed, Elseviers Science Publishers, NewYork, N.Y.). For viral replication kinetic determination (single stepgrowth curves), primary cultures of rat hippocampal neurons at day 6after plating were infected at a multiplicity of infection of 10(MOI=10). After an adsorption phase of 1 hour, virus was removed and theinitial media (MEM, 10% horse serum, 2 mM glutamine) was added to theneurons, followed by incubation at 36 degrees C., 10% CO₂, At theindicated times after infection, cells were collected by gentle scrapingand centrifuged. The resulting pellet was re-suspended in 1 ml ofsupernatant. Seven cycles of freezing and thawing were performed beforedetermining the viral titers. Titers were expressed in plaque formingunits (PFU) per ml (PFU/ml). Single step growth curves were obtained byplotting titer versus time on a logarithmic axis.

[0158] Antibodies

[0159] The following antibodies were used in these studies: anti-GFAP(glial fibrillary acidic protein) and anti-GalC (galactocerebroside)antibodies were part of the Neural Cell Typing Set for Identificationand Typing of Neural Cells (neurons, astrocytes, oligodendrocytes; RocheMolecular Biochemicals, Indianapolis, Ind.); anti-tubulin J (P.Yarowsky, University of Md., Baltimore); p-Erl (P-MAPK), whichrecognizes both phosphorylated forms of Erkc (MAPI) enzyme (p42 and p⁴4)(Promega, Madison, Wis.); anti-PARP (Roche Molecular Diagnostics,Indianapolis, Ind.) caspase-3 antibody (Santa Cruz Biotechnology, SantaCruz, Calif.).

[0160] Treatment with Apoptosis Inducers

[0161] Apoptosis was induced in HEI293 and JHLA1 cells usingstaurosporine (STS; Calbiochem, San Diego, Calif.) and D-manritol(D-Mann; Sigma, St. Louis, Mo.). Cells were plated in 6-well plates onglass coverslips or in flasks and allowed to grow for 24 hours at 37° C.in DMEM with 10% FBS, 1 mM sodium pyruvate, 0.1 mM non-essential aminoacids, and antibiotics. At 24 hours after plating, media was removed byaspiration, and appropriate dilutions of apoptotic inducers (STS 250 nM,D-Mann 300 mM) were added to the cells for various times indicated inthe text. All dilutions were made in cell growth media described abovecontaining 1% FBS. At the indicated times after treatment, cells wereprocessed for TUNEL or harvested for DNA extraction or Western blotassay.

[0162] TUNEL (TdT-Mediated dUTP Nick End Labeling)

[0163] In situ analysis of apoptosis was performed using In Situ CellDeath Detection Kit (Roche Molecular Biochemicals, Indianapolis, Ind.)according to the manufacturer's instructions. Briefly, infected/treatedor non-infected/non-treated cells were fixed with 4% paraformaldehyde(PFA) in PBS (pH=7.4) for 30 minutes at room temperature followed bypermiabilization of cells in 0.1% Triton X-100 (in 0.1% sodium citrate)for 2 minutes on ice. DNA breaks were labeled by addition of terminaldeoxynucleotidyl transferase (TdT) and nucleotide mixture (containingdUTP-fluorescein conjugated) and incubation for 60 minutes at 37° C.Coverslips were mounted in PBS/glycerol, and cells were analyzed byfluorescence microscopy. After extensive washes in PBS, cells wereincubated for 30 minutes at 37° C. with an anti-fluorescein antibodyconjugated with alkaline phosphatase (AP). Chromogenic reaction wascarried out by adding AP substrate solution (NBT/BCIP in 0.1 M Tris-HClpH=9.5, 0.05 M MCl₂, 0.1M NaCl, and 1 mM levamisole) and incubation for10 minutes at room temperature. Coverslips were mounted in PBS/glyceroland analyzed by light microscopy. Apoptotic cells (characterized by adark precipitate) and non-apoptotic cells (diffuse, light staining) werecounted in at least five randomly chose microscopic fields (containingat least 250 cells). The percentage of mock-infected neurons undergoingapoptosis (between 4 and 5%) was subtracted from each average.

[0164] Apoptotic index refers to the average percentage ofTUNEL-positive cells +SEM4.

[0165] Double labeling TUNEL/Neuronal Markers

[0166] Infected or non-infected hippocampal cultures were fixed with 4%PFA in PBS (pH=7.4) followed by permeabilization in 0.1% Triton X-100(in 0.1% sodium citrate). Cells were incubated with TdT and nucleotidemixture (containing dUTP-fluorescein conjugated) for 1 hour at 37° C.,followed by immunostaining with appropriate neuronal markers(anti-tubulin beta, anti-GFAP, anti-GalC). Signal was detected byincubating the cells with anti-mouse Ig conjugated to phycoerythrine(1:50 dilution) for 30 minutes at room temperature. Coverslips weremounted in PBS/glycerol and analyzed by fluorescence microscopy (CarlZeiss microscope) using the appropriate filters.

[0167] DNA Fragmentation Assay

[0168] HEK293, JHLA1 and JHL15 cells treated or not treated withapoptotic inducers were collected by gentle scraping at 24 hourspost-treatment/infection. DNA was extracted using Puregene DNA IsolationKit (Gentra Systems, Minneapolis, Minn.) according to the manufacturer'sinstructions. After spectrophotometrical quantitation, 5-10 microgramsof DNA per samples was separated on a 1.5% agarose gel stained with 0.1microgram/ml ethidium bromide. Fragmented DNA was visualized by exposureto UV light and photographed using a Polaroid MP-4 land camera.

[0169] Western Blot Assay

[0170] Cells were collected by gentle scraping and centrifuged at 2,500rpm for 5 minutes at 4° C. The dry pellet was stored at −80° C. untiluse. Cells were lysed with RIPA buffer (30 mM Tris-Hcl pH=7.4, 0.15 mMNaCl, 1% Nonidet P-40, 0/1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1mM DTT, 2 mM MgCl₂, 0.5 mM PMSF) supplemented with phosphatase andprotease inhibitor cocktails (Sigma, St. Louis, Mo.) and sonicated for 1minute at 25% output power using the Sonicator/Ultrasonic Processor(Misonix, Inc., Farmingdale, N.Y.). Total protein was determined by BCA(Pierce, Rockford, Ill.) and proteins were resolved by SDS-PAGE.Transfer of separated proteins to Schlecher and Schuell nitrocellulosemembranes was performed overnight using an electroblotting apparatus.Non-specific binding was blocked by incubating blots for 1 hour at 37°C. in TN-T buffer (0.01 M Tris-Hcl pH=7.4, 0.15 M NaCl, 0.05% Tween 20)containing 1% bovine serum albumin (BSA). After washing with TN-Tbuffer, the blots were incubated for 2 hours at room temperature withthe appropriate antibodies diluted in TN-T buffer containing 0.1% BSA.After three washes with TN-T buffer, the blots were incubated withProtein A-Peroxidase for 1 hour at room temperature. Detection wasperformed using ECL reagents (Amersham Life Science, Arlington Heights,Ill.) and exposing the blots to high performance chemiluminescence film(Hyperfilm ECL, Amersham Life Science, Arlington Heights, Ill.).

[0171] It has been shown previously that the Ras/MEK/MAPK mitogenicpathway is activated in cells that express ICP10PK (JnLa1), but not itsPK negative mutant, p138TM (JHL15) (Smith et al., 1994, Virology,200:598-612). Because this pathway was implicated in the control ofapoptosis (Kaplan et al., 2000, Curr. Opin. Neurobiol., 10:381-391), theresponse of JHLa1, JIL15 and parental (HEK293) cells to the apoptosisinducers STS and D-Mann was examined. HE1K293, JHLA1, and JHL15 cellswere treated for 24 hours with 250 nM STS or 300 nM D-Mann and apoptoticcells were quantitated using the TUNEL assay. Results of threeindependent experiments are expressed as percentage of apoptotic cells±SEM. The proportion of TUNEL-positive cells (apoptotic) wassignificantly higher is STS-treated HEK293 (71±12.4%) and RHL15(79±4.9%) than JHLA15 (5.8+2.1%) cells, and similar results wereobtained in D-Mann-treated cells (79±7.4%, 58±7%, and 13±8% for HEK293,JHL15, and JHLA15 cells, respectively) (p<0.01 by Student t test) (FIG.1A). STS-treated, TUNEL-positive HEK293 and JHL15 cells evidenced thehallmark morphological features of apoptosis including cell shrinkage,condensed chromatin, and nuclear fragmentation (FIGS. 1B and 1C), butthese features were not observed in the STS-treated, TUNEL negativeJHLA1 cells (FIG. 1D). The data indicate that ICP10PK has anti-apoptoticactivity, since the only difference between JHLA1 and JHL15 cells is thePK activity of ICP10.

[0172] To determine whether ICP10PK blocks DNA fragmentation induced bySTS and D-Mann, treated cells were analyzed using a DNA fragmentationassay which detects degradation of chromosomal DNA into oligonucleosomalfragments. HEK293 cells treated with STS or D-Mann exhibited a DNAladder pattern characteristic of apoptosis (FIG. 2, lanes 3 and 7,respectively), and similar results were obtained in JHL15 treated cells.In contrast, mock treated HEK293 cells (FIG. 2, lanes 2 and 6) and JHLAIcells treated with STS or D-Mann (FIG. 2, lanes 4 and 8, respectively)did not exhibit DNA fragmentation. These results indicate that ICP10PKblocks fragmentation of cellular DNA induced by apoptotic stimuli.

[0173] Previous reports have shown that STS-induced cell death is acaspase-dependent process (Jacobson et al., 1996, J. Cell. Biol.,133:1041-1051). Cleavage of the inactive procaspases (e.g. procaspase-3)causes their activation, and this is a central determinant of manyapoptotic processes. To determine whether ICP10PK inhibits cleavage ofpro-caspase-3, a central determinant of many apoptotic processes,Western blot analysis was performed on cell lysates obtained fromSTS-treated cells. A 32 kDa band consistent with the inactiveprocaspase-3 (caspase-3p32) was detected by immunoblotting withcaspase-3p32 antibody in HEK293, JHL15, and JHLA1 cells treated withDMSO (STS diluent) or STS (250 nM). However, densitometric scanningindicated that levels of caspase-3p32 were significantly lower in HEK293and JBL15 cells treated with STS (FIG. 3A, lanes 2 and 4) than DMSO(FIG. 3A, lanes 1 and 3). The densitometric units were equal to 0.7 forboth STS-treated HEK293 and JHL15 cells and 1.0 and 1.1 for DMSO-treatedHEK293 and JHL15 cells, respectively). The levels of caspase-3p32 weresimilar in STS-treated and untreated JHLA1 cells (FIG. 3A, lanes 5 and6, respectively). In this case, the densitometric units were equal to1.2 for both treatments. Conversely, the mean percentage of cellspositive for the activated caaspase-3 cleavage product (caspase-3p20)was significantly p<0.01 by Student t test) higher in STS-treated HEK293(40.5±0.9%) and JHL15 (36.4±7.6%) than in STS-treated JHLA1 (9.3±0.1%)cells, as determined by immunohistochemistry with antibody specific forthis product (Cell Signaling Technologies, Beverly, Mass.) (FIG. 3B).The data indicate that ICP10PK interferes with caspase-3p32cleavage/activation.

[0174] To determine whether ICP10PK blocks PARP cleavage by caspase-3, ahallmark of caspase-3 dependent apoptosis, Western blot analysis usingPARP antibody was performed on STS-treated HEK293, JHL15, and JHLA1cells. A 116 kDa band consistent with the uncleaved PARP was detected inmock- and STS-treated cells. In contrast, an 85 kDa band consistent withthe PARP cleavage product was detected only in STS-treated HEK293 andJHL15 cells (FIG. 3C, lanes 2 and 4, respectively), but not inSTS-treated JHLA1 cells (FIG. 3C, lane 6)or mock-treated HEK293, JHL15,JHLA1 cells (FIG. 3C, lanes 1,3, and 5, respectively). These datafurther suggest that 1CPlOPK blocks caspase-3 dependent apoptosis.

[0175] Taken together, the data presented above demonstrate: (i) ICP10protects cells against apoptosis induced by STS and D-Mann, suggestingthat ICP10 acts on a downstream effector molecule in common to theapoptotic pathway stimulated by these inducers, (ii) ICP10PK inhibitscaspase-3 dependent apoptosis, as determined by its ability to inhibitcleavage of procaspase-3 and PARP. However, the ability of ICP10 toinhibit apoptosis is not restricted to caspase-3 and it may involveinhibition of the activation of other caspases as well as the activationof anti-apoptotic proteins involved in the death pathway.

[0176] To determine whether apoptosis or protection thereof is relatedto virus replication in hippocampal neurons, primary rat hippocampalcultures (E17-18) were infected with HSV-2, ICP10deltaPK, orICP10deltaRR (shown schematically in FIG. 4A), and the replicationkinetics were evaluated. Briefly, hippocampal neurons at day six inculture were infected with 10 PFU/cell and virus titers were determinedby plaque assay at the indicated times post-infection (pi) (FIG. 4B).HSV-2 and HSV-1 replicated equally well in these cells, with growthcurves similar to those previously described for other cell types (Smithet al., 1998, J. Virol., 72:9131-9141). Replication began at 3-5 hoursp.i. reaching maximal titers at 15-24 hours p.i. ICP10deltaPK andICP10deltaRR were growth-defective in these cells. These data suggestthat ICP10 is required for virus growth.

[0177] Because anti-apoptotic activity is often cell-type specific, thefollowing series of experiments were designed to test whether ICP10PKhas anti-apoptotic activity during viral infection of hippocampalneurons. Cells were infected with 10 PFU/cell of HSV-2 or ICP10deltaPKand apoptosis was determined by TUNEL at 16 and 24 hours p.i. A total of200-300 cells were counted in five randomly chosen microscopic fieldsand the mean percentage of TUNEL-positive cells was determined. The datashown in FIG. 5A represent the results of three independent experiments.The percentage of apoptotic cells was significantly lower in culturesinfected with HSV-2 than ICP10deltaPK (5.7±0.5% and 68±3.9%,respectively; p<0.01 by Student t test). Apoptosis was alsosignificantly lower in cultures infected with ICP10deltaRR (whichretains ICP10PK DNA) than ICP10deltaPK (23±2.1% and 68±3.9%respectively: p°0.01 by Student test). Similar results were obtained at16 hours p.i. (% apoptotic celss=44±3.5% and 5.4±1.2% for ICP10dletaPKand HSV-2, respectively). The percentage of ICP10deltaPK infected cellsthat underwent apoptosis increased with increasing multiplicity ofinfection (2S.6±4.9% and 68±3.9% for 1 and 10 PFU/cell, respectively),but apoptosis was equally low in cultures infected with HSV-2 at bothmultiplicities if infection (4.8±1.1% and 5.7±0.5% for 1 and 10PFU/cell, respectively. Staining with the fluorescent DNA-binding dyeHoechst 32258 revealed the presence of nuclear fragmentationcharacteristic of apoptosis in ICP10deltaPK but not HSV-2-infected cells(FIGS. 5B and 5D, respectively). The data indicate that ICP10PK hasanti-apoptotic activity in hippocampal neurons.

[0178] Because ICP10PK has limited structure and functional similarityto its HSV-1 counterpart (also known as ICP6PK), we investigated whetherICP6PK also has anti-apoptotic activity in cultures of hippocampalneurons. Cell cultures were infected with HSV-1, HSV-2, ICP10deltaPK, orICP10deltaRR at 10 PFU/cell and assayed by TUNEL at 24 hours p.i. Asimilar percentage of TUNEL-positive cells was seen for HSV-1(50.2±4.5%) and ICP10deltaPK (68±3.9%) (p>0;05 by Student t test) (FIG.5A). This percentage was significantly higher (p<0.01 by Student t test)than that seen for HSV-2 (5.7±0.5%) or ICP10deltaRR (23+2.1%)HSV-1-infected hippocampal neurons also evidenced nuclear fragmentationpatters characteristic of apoptosis (FIG. 5C).

[0179] Since TUNEL may be an unreliable assay due to label incorporationat sites of random DNA degradation and the results obtained withconstitutively expressing cells indicated that ICP 10PK blocks caspase-3dependent apoptosis, caspase-3 activation in hippocampal cultures wasanalyzed. Cultures were infected for 24 hours with HSV-2, ICP10deltaPK,ICP10deltaRR, or HSV-1 at 10 PFU/cell and stained with caspase-3p20antibody. The percentage of caspase-3p20-positive cells wassignificantly higher in cultures infected with ICP10deltaPK (52.6±9.6%)or HSV-1 (47.4±%) than HSV-2 (19.3±2%) or ICP10deltaRR (26±0.2%) whichretains ICP10PK DNA (p<0.05 by Student t test) (FIG. 5E). The data arecomparable to those obtained by TUNEL, supporting the conclusion thatICP10PK, but not its HSV-1 counterpart (ICP6PK), has anti-apoptoticactivity in primary cultures of hippocampal cells.

[0180] The primary hippocampal cultures used in these studies are likelyto consist of various cell subpopulations. Therefore, to identify thecells type(s) that become apoptotic upon infection with ICP10deltaPK orHSV-1, cell cultures were infected for 24 hours with ICPlOdeltaPK orHSV-I at 10 PFU/cells. Cells were double stained with fluorescein(FITC)-labeled dUTP (TLINEL) and phycoerthlrine (PE)-labeled antibodiesspecific for different cell subpopulations. TUNEL-positive cells wereobserved in cultures infected with ICP10deltaPK (FIG. 6A) or HSV-1 (FIG.7D) and also stained with TUJ-1 antibody (FIGS. 6B and 6E) which isspecific for postmitotic neurons (Fereira et al., 1992, J. Neurosci.Res., 32:516-529). The TUJ-1 staining (PE) localized in the cell bodiesand neurites while the FITC staining (TUNEL) was nuclear (FIGS. 6C and6F). TUNEL-positive cells did not stain with glial fibrillary acidicprotein (GFAP) or galactocerebroside (GalC) antibodies that are specificfor astrocytes and oligodendrocytes, respectively. The data indicatethat the apoptotic cells are neurons. By inference, ICP10PK, but notICP6PK, protects hippocampal neurons against apoptotic stimulus.

[0181] Having shown that ICP10PK has anti-apoptotic activity inhippocampal neurons, it was of interest to determine whether thisactivity is related to its ability to activate the MEK/MAPK pathway(Smith et al., 1994, Virology, 200:598-612; Smith et al., 2000, J.Virol., 74:10417-10429). Culture of hippocampal neurons were infectedwith HSV-2 or ICP10deltaPK at 10 PFU/cell and analyzed for MAPKactivation by immunoblotting with antibodies specific for theunphosphorylated (MAPK) and phosphorylated (activated) MAPK species(P-MAPK1/2). HSV-1 was used as a control because (i) it does notactivate MEK/MAPK (McLean et al., 1999, J. Virol., 73:8415-8426; Zachoset al., 1999, J. Biol. Chem., 274:5097-5103) and (ii) ICP6PK does nothave anti-apoptotic activity. Cell extracts were studies at 0.5 and 24hours p.i. (0 hours p.i. is at the end of adsorption). At 0.5 hoursp.i., P-MAPK1/2 levels were significantly higher in HSV-2-infected (FIG.7A, lane 2) than mock-infected (FIG. 7A, lane 1) cells. The increasedlevels of P-MAPK1/2 in HSV-2 infected cells reflect MAPK activation byICP10PK because (i) P-MAPK1/2 levels were not increased in cellsinfected with ICP10deltaPK (FIG. 7A, lane 2) or HSV-1 (FIG. 7A, lane 4)and (ii) the levels of the unphosphorylated MAPK species were similar inall cultures (FIG. 7A, bottom). MAPK activation was not observed at 24hours p.i., and similar P-MAPK1/2 levels were observel in all cultures(FIG.7A. lanes 5-7). Densitometric scanning of the bands in FIG. 8Asupport these conclusions (FIG. 7B).

[0182] To examine the contribution of upstream components of the Rassurvival pathway towards MIAPK activation, hippocamnpal cultures wereinfected with 5 HSV-2 in the presence (or absence) of 20 microiioles ofthe MEK-specific inhibitor UO126 and cell extracts were examined forP-MAPK1/2 by inmuiunoblotting wvith specific antibody. P-MAPK1/2 levelswere significantly lower in cells treated with UO126 (FIG. 7C, lanes 2and 3; FIG. 7D) than in untreated cells (FIG. 7C,. lane 2; FIG. 7D),suggesting that MPAK activation is MEK dependent. Presumably MEK/MAPKactivation is responsible for the anti-apoptotic activity of HSV-2,since U0126 treatment of HSV-2 infected cultures caused a dose-dependentincreased in the percentage of TUNEL-positive (apoptotic) cells (20±1.6%and 39.3±2.8% for 10 and 20 micromoles, respectively, as compared to7.2±0.7% in untreated cells; p<0.01 by Student t test) (FIG. 7E).

[0183] The possibility that P13-kinase activation is also involved inthe anti-apoptotic activity of HSV-2 was considered, because theP13-kinase/Akt pathway functions in growth factor initiated survival inneurons (Kaplan et al., 2000, Curr. Opin. Neurobiol., 10:381-391).However, the percentage of TUNEL-positive cells in hippocampal culturesinfected with HSV-2 in the presence of LY294002 [at concentrations(10-100 micromolar) at which it specifically inhibits P13-kinase](Crowder et al., 1998, J. Neurosci., 18:2933-2943) were only minimallyincreased [from 9.1±1 (untreated) to 24.8±2.3 (when treated with 100micromolar LY294002)] and died more slowly (p<0.01 by ANOVA) than themock-infected cells treated with inhibitor [5±3 (non-treated) to 72±4.5(when treated with 100 micromolar LY294002)] (FIG. 7F). Same kineticswere observed when cells were treated with wortmannin (Yano et al.,1998, Nature, 396:584-587) at concentrations (100-200 nanomolar) thatspecifically inhibit PI3-K (data not shown). These data suggest thatPI3-K has only minimal contribution to the survival of HSV-2 infectedhippocampal neurons, but it is however required for the basalmaintenance of these cells, at least under the present experimentalconditions. v The finding that MEK/MAPK are activated in hippocampalcells at 30 minutes, but not 24 hours p.i. is amenable to two potentialinterpretations. According to the first interpretation, MEK/MAPKactivation (and, therefore, anti-apoptotic activity is mediated by theICP10PK located in the tegument of the incoming virion (Smith et al.,1997, Virology, 234:235-242). Implicit in this interpretation is theassumption that cellular penetration and virion Lncoatihi; are requiredfor both processes. An alternative interpretation is that MEK/MAPK areactivated by virus binding to receptors on the cell surface, and both itand the resulting anti-apoptotic effect are independent of cellpenetration. This possibility is particularly significant because theHSV receptor can generate a signal that regulates AP-1 upon ligandbinding (Marsters et al., 1997, J. Biol. Chef., 272:14029-12032). Todetermine whether MEK/MAPK activation is dependent on cellularpenetration, cultures of hippocampal neurons were infected withantibody-neutralized HSV-2 that binds, but does not penetrate the cells(Highlander et al., 1987, J. Virol., 61:3356-3364). Cells were examinedfor MAPK activation by immunoblotting with antibody specific forP-MAPK1/2 at 30 minutes p.i. P-MARK1/2 levels were significantly lowerin cells exposed to virus neutralized with a monoclonal antibody toglycoprotein D (gD Mab) (FIG. 8A, lane 3; FIG. 8B) or HSV-2 antiserum(FIG. 8C, lane 3; FIG. 5D) than non-neutralized virus (FIG. 5A, lane 2;FIG. 8B) or virus neutralized with pre-immune serum (FIG. 5C, lane 2;FIG. 5D). The superior effect of the anti-HSV-2 serum relative to the gDMab presumably reflects the broader antigenic specificity of theantiserum. These data suggest that cell penetration is required forMEK/MAPK activation.

[0184] Implicit in the assumption that cell penetration is required forMEK/MAPK activation and anti-apoptotic activity is the conclusion thatboth are mediated by the ICP10 PK located within the virion tegument,which is released upon virion uncoating (Smith et al., 1997, Virology,234:235-242). To test this hypothesis, extracts of cells infected withHSV-2, ICP10deltaPK, ICP10deltaRR or neutralized HSV-2 were obtained at30 minutes p.i. and immunoblotted with antibody that recognizes ICP10and its mutants (p95 and p175 for ICP10deltaPK and ICP10deltaRR,respectively). ICP10 (FIG. 9, lane 3), p95 (FIG. 9, lane 4) and p175(FIG. 9, lane 5) were observed in extracts of cells infected with HSV-2,ICP10deltaPK and ICP10deltaRR. However, these proteins were not observedin extracts from cells infected with neutralized HSV-2 (FIG. 9, lane 2),consistent with the failure of the neutralized virus to penetrate thecells (Highlander et al., 1987, J. Virol., 61:3356-3364). The dataindicate that MEK/MAPK activation and anti-apoptotic activity aremediated by the virion ICP10Pk and require cell penetration/virionuncoating.

Example 2 Tle Use of lISV-2 ICP10PK to Treat Neurodeoenerative Disorders

[0185] The data herein demonstrate the ability of ICP10PK to protectcells from apoptotic stimuli specific for neurodegenerative disorderssuch as growth factor deprivation and genetic defects. Also demonstratedherein is delivery of ICP10PK to the CNS upon non-invasive, peripheraladministration, using a viral vector.

[0186] The materials and methods used in the experiments presented inthis example are now described.

[0187] Transfection of PC12 Cells

[0188] PC12 cells were cultured in DMEM/F12 (Gibco-BRL) with 10% fetalbovine serum (FBS) (Gemini), 0.36% D-giucose (Sigmna), 0.21% sodiumbicarbonate, 0.009% gentamycin and 100 ng/ml NGF (Roche MolecularBiochemicals) and transfected [using FuGene 6 Transfection Reagent(Roche Molecular Biochemicals)] with vectors pJW17 or pJHL15 thatrespectively express ICP10 or a TM deleted PK negative ICP10 mutant(p139™) (Luo et al., 1992, J. Biol. Chem., 267:9645-53; Smith et al.,1994, Virology, 200:598-612). At 24 hours post-transfection, the cellswere washed and the medium was replaced with medium free of NGF.

[0189] Cell Viability Assay

[0190] Cell viability was determined using CellTiter 96 Aqueous OneSolution Cell Proliferation Assay (Promega) that measures enzymaticactivity of functional mitochondria, according to the manufacturerinstructions.

[0191] The next series of experiments were designed to test thetherapeutic value of ICP10PK in treating neurodegenerative disorders.Thus, the effect of ICP10 PK on: i) neuronal apoptosis induced byapoptosis stimulus specific for these neurological conditions (loss oftrophic support), and ii) accelerated neuronal death of trisomy 16 (anaturally occurring mouse genetic disorder) hippocampal neurons wastested.

[0192] Because Ras/MEK/MAPK is involved in neurotrophin-mediatedneuronal survival, it was of interest to determine whether ICP10 PK mayalso promote survival in experimental paradigms created by trophicfactor deprivation or genetic defects in neurotrophin receptorsignaling. In a first series of experiments rat pheochromocytoma (PC12)cells were used as an in vitro model to test this hypothesis. Whengrowth in NGF containing medium, PC12 cells acquire properties ofsympathetic neurons (neurite outgrowth, electrical excitability andexpression of specific neuronal markers) and die by apoptosis upon NGFvwithdrawal (Greene et al., 1982, Adv,. Cell. Neurobiol., 3:373-414).Because these cells can be rescued by the addition of NGF (Greene etal., 198S, Adv. Cell. Neurobiol., 3:373-414), the ability of 1CP10PK tosubstitute for NGF with respect to its ability to support survival ofdifferentiated PC12 cells was evaluated. PC12 cells were transfectedwith vectors encoding ICP10 and p139™, and ICP10 and p139™ expressionwas examined at 24 hours post-transfection by straining with anICP10-specific antibody (recognizes amino acids 13-26 in both proteins),as previously described (Aurelian et al., 1989, Cancer Cells,7:187-191).

[0193] Expression of the transfected genes was detected in approximately35-50% of the cells. Staining was localized in the cytoplasm and bothits intensity and the proportion of stained cells were similar for pJW17(FIG. 10B) and pJHL15 (FIG. 10C), suggesting that ICP10 and p139™ areexpressed equally well. Staining was specific, and was not seen incontrol (non-transfected) PC 12 cells (FIG. 10A). Morphologically, thenon-transfected PC12 cells (FIG. 10A) exhibited degenerated cell bodiesand “beading” of neurites starting at approximately 24 hours after NGFremoval, while the pJW17-transfected cultures were debris-free, withlong neurites and cell bodies resembling those of NGF-treated PC12cells.

[0194] Cell viability was determined at 24, 48 and 72 hourspost-transfection (0, 24 and 48 hours post-NGF withdrawal). Results areexpressed as % viable cells ±SEM relative to 0 hours after NGFwithdrawal. The kinetics of cell death in the non-transfected andpJHL15-transfected cells were similar to those previously reported forthis system (Pittman et al. 1993), with a respective survival of65.7±2.7% and 61.2±1% at 24 hours after NGF withdrawal and 48.1±1% and43.1±1.4% at 48 hours after NGF withdrawal (FIG. 11). By contrast,survival of pJHL17-transfected cells was 82.3±3.9% and 73.2±3.3% at 24and 48 hours after NGF removal (p<0.05 vs. control and pJBL15transfected cells, by ANOVA), suggesting that ICP10 PK can compensatefor the absence of NGF.

[0195] To further examine the role of ICP10PK in neuronal survival, theexperiment was repeated with primary hippocampal culture from embryonicday 16 (E16) mice, established and grown as previously described(Bambrick and Krueger, 1999) on glass coverslips etched with a grid of175×175 micrometer squares (CELLocate; Eppendorf) in MEM with B27supplement (Gibco) which contain optimized concentrations of neuronsurvival factors. At 2 days in culture, the cells were transfected withpJW 17 or pJl-L 15 and the medium was replaced with NEM free of serumand growth factors (O hours) as described above. Control nion- Stransfected cultures were maintained in medium with (Eu+B27) or without(Eu-B 27) the B27 supplement and neuronal survival was determined bycounting live cells (phase-dark bodies and fine neurites) in sevenrandomly chosen squares (Bamblick et al., 1999, J. Neurochem.,72:1769-1772). Neuronal identity was confirmed by staining with theneuron-specific antibody to beta tubulin (TuJ1) (Ferreira et al., 1992,J. Neurosci. Res., 32:516-529). The results are expressed as percentageof surviving cells±SEM relative to 0 hours. The % surviving cells wererespectively 52.6±7.2% and 56±3.1% for Eu-B27 and pJHL 15-transfectedcells at 48 hours and 34.3±7.6% and 34.8±3.8% at 72 hours after B27withdrawal. The viability of pJW17-transfected cells was significantly(p<0.05 by ANOVA) higher (80.6±2.7% and 67.7±2.4% at 48 and 72 hours,respectively) and similar to that of Eu+B27 cells (88.7+3.1% and80.6+2.7% at 48 and 72 hours, respectively) (FIG. 12). These data areconsistent with those obtained for differentiated PC12 cells andindicate that ICP10PK also promotes the survival of hippocampal neuronsin the absence of growth factors.

[0196] The trisomy 16 (Tsl6) mouse is considered to be a model of Down'ssyndrome (DS; trisomy 21) (Coyle et al., 1988, Trends Neurosci.,11:390-394) with a cluster of genes and loci on chromosome 16 that arealso located on the long arm of human chromosome 21 (Sawa et al., 1999,J. Neural Transm [Suppl], 57:87-87). DS individuals develop Alzheimer'sdisease (AD) by their fifth decade (Sawa et al., 1999, J. Neural Transm[Suppl], 57:87-87), suggesting that this genetic defect also conferincreased vulnerability to neurodegeneration. Cultured hippocampalneurons from Ts16 mouse exhibit augmented cell death when compared toeupoid cells even in the presence of adequate trophic support (Bambricket al., 1999, J. Neurochem., 72:1769-1772). Accordingly, in this seriesof experiments designed to examine whether ICP10PK can block apoptosisin neuronal cells, we used primary hippocampal cultures from Ts16 mice,established as described (Bambrick et al., 1999, J. Neurochem.,72:1769-1772). The cells were transfected with pJW17 or pJHL15 at twodays in culture and maintained in B27-supplemented medium for theduration of the experiment. Non-transfected (Ts16) neurons evidenced anaccelerated death rate (76.2-.3,. *37_(—)7 28, ;ii1 41.4 .3”;- survivalit 24, 4S algad 72 hours, respectively) relative to euploid neurons(Eu-+B7) (93.S+1 .7”/o, 88.7±3.1%, (mdl S0.6+2.7% survival at 24, 48,and 72 hours, respectively) (pI<.Ol by ANOVA) (FIGS. 13 and 14A).Simnilir cell deaths kiietics wele seen for pfILl5-traiisfected Tsl6netrons (73.8±3.0%, 54.9+3.2% and 43.8±3.7%, at 24, 4S, and 72 hours,respectively) (p>0.05 vs. non-transfected Tsl6 neurons by ANOVA) (Fiutre13 and 14B). By contrast, the survival of pJW17-transfected Ts16 neurons(93.5±1.5%, 83.9±2%, and 75.6±2.2% at 24, 48 and 72 hours (p>0.05 vs.non-transfected Ts16 neurons (93.5±1.5%, 83.9±2%, and 75.6±2.2% at 24,48 and 72 hours, respectively) was similar to that of Eu+B27 cells andsignificantly (p<0.001., by ANOVA) increased than that ofnon-transfected Ts16 neurons (FIGS. 13 and 14C). The mechanism of Ts16cell death was examined by TdT-medicated dUTP nick end labeling (TUNEL),an assay that is widely considered to be specific for apoptosis(Gavrieli et al., 1992, J. Cell. Biol., 119:493-501). Ts16 cultures(non-transfected or transfected with pJW17 or pJHL15 expression vectors)were fixed with 4% paraformaldehyde at day five in vitro (72 hourspost-transfection) and TUNEL was carried out using the In Situ CellDeath Detection Kit-AP (Roche Molecular Biochemicals) according to themanufacturer instructions. Apoptotic cells (characterized by a darknuclear precipitate ) and non-apoptotic cells (unstained or displaying adiffuse, light and uneven cytoplasmic staining) were counted in fiverandomly chosen microscopic fields (containing at least 250 cells).Results are expressed as percentage of apoptotic cells±SEM. Theproportion of TUNEL-positive cells (apoptotic) in non-transfected(43±3.4%) and pJHL15-transfected (39±1%) correlate well (within thelimits of experimental error) with the percentage of dead cells obtainedby counting the morphologically viable cells (approximately 60% death atday 5 in vitro or 72 hours post-transfection). The difference inabsolute values may be due to the loss of some cells during the fixationprocedure and/or the apoptotic process itself. By contrast, thepercentage of apoptotic cells was significantly lower (p<0.001, byANOVA) in pJW17-transfected Ts16 cells (9.4±1.1%) similar to thatobtained for euploid cultures maintained in MEM with B27 supplement(Eu±B27) (9.6±2%) (FIG. 15). These data indicate that ICP10PK blocks theapoptotic death of Ts16 neurons. Similar results were obtained in threeindependent experiments.

[0197] To examine whether the anti-apoptotic protein 1CP10PK can bedelivered to the CNS Using the ICPl0deltl?JR vector, the natural routeof transmission of HSV to the CNS by means of olfactory nerves andtracts was mimicked. Mice were infected intraniasally with variousamounts of ICP10deltaRR virus or phosphate buffer saline (as a control)and sacrificed one week later. Coronal sections of the brains werein-ununostained with antibody specific for ICP10 (FIG. 16). ICP10deltaRRvirus is growth comlproliiscd in vitro and in vivo followinginoculation. Significantly, the intranasally infected mice were free ofneurological impairments or other untoward effects associated with HSV-2infection. Data suggest that peripheral (intranasal) administration ofICP10OΔPK with the CP10deltaRR vector induces protein expression atleast up to one week in hippocampus and related limbic structures.

Example 3 A bcl-2-Expressing HSV-2 ICP10deltaRR Mutant Virus

[0198] To construct the ICP10deltaRR mutant virus, the LacZ coding genein the ICP10deltaRR plasmid is replaced with the gene encoding bcl-2,and the virus is rescued by recombination screening for white plaques.The strategy is similar to that which was used to construct theICP10deltaPK virus (Smith et al., 1998, J. Virol., 72:9131-9141; Peng etal., 1996, Virology, 216:184-196).

[0199] While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1 2 1 5956 DNA herpes simplex virus type 2 1 gtgtgtttgg cgtgtgtctctgaaatggcg gaaaccgaca tgcaaatggg attcatggac 60 acgttacacc cccctgactcaggagatagg catatcctcc ttagattgac tcagcacacg 120 atcgcacccc acccctgtgtgccggggata aaagccaacg cgggcggtct gggttaccac 180 aacaggtggg tgcttcggggacttgacggt cgccactctc ctgcgagccc tcacgtcttc 240 gcccaccgat tcctgttgcgttcctgtcgg ccggtgctgt cctgtcgaca gattgttggc 300 gactgcccgg gtgattcgtcggccggtgcg tcctttcggt cgtaccgccc accccgcctc 360 ccacgggccc gccgctgtttccgttcatcg cgtccgagcc accgtcacct tggttccaat 420 ggccaaccgc cctgccgcatccgccctcgc cggagcgcgg tctccgtccg aacgacagga 480 accccgggag cccgaggtcgccccccctgg cggcgaccac gtgttttgca ggaaagtcag 540 cggcgtgatg gtgctttccagcgatccccc cggccccgcg gcctaccgca ttagcgacag 600 cagctttgtt caatgcggctccaactgcag tatgataatc gacggagacg tggcgcgcgg 660 tcatttgcgt gacctcgagggcgctacgtc caccggcgcc ttcgtcgcga tctcaaacgt 720 cgcagccggc ggggatggccgaaccgccgt cgtggcgctc ggcggaacct cgggcccgtc 780 cgcgactaca tccgtggggacccagacgtc cggggagttc ctccacggga acccaaggac 840 ccccgaaccc caaggaccccaggctgtccc cccgccccct cctcccccct ttccatgggg 900 ccacgagtgc tgcgcccgtcgcgatgccag gggcggcgcc gagaaggacg tcggggccgc 960 ggagtcatgg tcagacggcccgtcgtccga ctccgaaacg gaggactcgg actcctcgga 1020 cgaggatacg ggctcgggttcggagacgct gtctcgatcc tcttcgatct gggccgcagg 1080 ggcgactgac gacgatgacagcgactccga ctcgcggtcg gacgactccg tgcagcccga 1140 cgttgtcgtt cgtcgcagatggagcgacgg ccctgccccc gtggcctttc ccaagccccg 1200 gcgccccggc gactcccccggaaaccccgg cctgggcgcc ggcaccgggc cgggctccgc 1260 gacggacccg cgcgcgtcggccgactccga ttccgcggcc cacgccgccg caccccaggc 1320 ggacgtggcg ccggttctggacagccagcc cactgtggga acggaccccg gctacccagt 1380 ccccctagaa ctcacgcccgagaacgcgga ggcggtggcg cggtttctgg gggacgccgt 1440 cgaccgcgag cccgcgctcatgctggagta cttctgtcgg tgcgcccgcg aggagagcaa 1500 gcgcgtgccc ccacgaaccttcggcagcgc cccccgcctc acggaggacg actttgggct 1560 cctgaactac gcgctcgctgagatgcgacg cctgtgcctg gaccttcccc cggtcccccc 1620 caacgcatac acgccctatcatctgaggga gtatgcgacg cggctggtta acgggttcaa 1680 acccctggtg cggcggtccgcccgcctgta tcgcatcctg gggattctgg ttcacctgcg 1740 catccgtacc cgggaggcctcctttgagga atggatgcgc tccaaggagg tggacctgga 1800 cttcgggctg acggaaaggcttcgcgaaca cgaggcccag ctaatgatcc tggcccaggc 1860 cctgaacccc tacgactgtctgatccacag caccccgaac acgctcgtcg agcgggggct 1920 gcagtcggcg ctgaagtacgaagagtttta cctcaagcgc ttcggcgggc actacatgga 1980 gtccgtcttc cagatgtacacccgcatcgc cgggttcctg gcgtgccggg cgacccgcgg 2040 catgcgccac atcgccctggggcgacaggg gtcgtggtgg gaaatgttca agttcttttt 2100 ccaccgcctc tacgaccaccagatcgtgcc gtccaccccc gccatgctga acctcggaac 2160 ccgcaactac tacacgtccagctgctacct ggtaaacccc caggccacca ctaaccaggc 2220 caccctccgg gccatcaccggcaacgtgag cgccatcctc gcccgcaacg ggggcatcgg 2280 gctgtgcatg caggcgttcaacgacgccag ccccggcacc gccagcatca tgccggccct 2340 gaaggtcctg gactccctggtggcggcgca caacaaacag agcacgcgcc ccaccggggc 2400 gtgcgtgtac ctggaaccctggcacagcga cgttcgggcc gtgctcagaa tgaagggcgt 2460 cctcgccggc gaggaggcccagcgctgcga caacatcttc agcgccctct ggatgccgga 2520 cctgttcttc aagcgcctgatccgccacct cgacggcgag aaaaacgtca cctggtccct 2580 gttcgaccgg gacaccagcatgtcgctcgc cgactttcac ggcgaggagt tcgagaagct 2640 gtacgagcac ctcgaggccatggggttcgg cgaaacgatc cccatccagg acctggcgta 2700 cgccatcgtg cgcagcgcggccaccaccgg aagccccttc atcatgttta aggacgcggt 2760 aaaccgccac tacatctacgacacgcaagg ggcggccatt gccggctcca acctctgcac 2820 ggagatcgtc cacccgtcctccaaacgctc cagcggggtc tgcaacctgg gcagcgtgaa 2880 tctggcccga tgcgtctcccggcggacgtt cgattttggc atgctccgcg acgccgtgca 2940 ggcgtgcgtg ctaatggttaatatcatgat agacagcacg ctgcagccga cgccccagtg 3000 cgcccgcggc cacgacaacctgcggtccat gggcattggc atgcagggcc tgcacacggc 3060 gtgcctgaag atgggcctggatctggagtc ggccgagttc cgggacctga acacacacat 3120 cgccgaggtg atgctgctcgcggccatgaa gaccagtaac gcgctgtgcg ttcgcggggc 3180 gcgtcccttc agccactttaagcgcagcat gtaccgggcc ggccgctttc actgggagcg 3240 cttttcgaac gccagcccgcggtacgaggg cgagtgggag atgctacgcc agagcatgat 3300 gaaacacggc ctgcgcaacagccagttcat cgcgctcatg cccaccgccg cctcggccca 3360 gatctcggac gtcagcgagggctttgcccc cctgttcacc aacctgttca gcaaggtgac 3420 cagggacggc gagacgctgcgccccaacac gctcttgctg aaggaactcg agcgcacgtt 3480 cggcgggaag cggctcctggacgcgatgga cgggctcgag gccaagcagt ggtctgtggc 3540 ccaggccctg ccttgcctggaccccgccca ccccctccgg cggttcaaga cggccttcga 3600 ctacgaccag gaactgctgatcgacctgtg tgcagaccgc gccccctatg ttgatcacag 3660 ccaatccatg actctgtatgtcacagagaa ggcggacggg acgctccccg cctccaccct 3720 ggtccgcctt ctcgtccacgcatataagcg cggcctgaag acggggatgt actactgcaa 3780 ggttcgcaag gcgaccaacagcggggtgtt cgccggcgac gacaacatcg tctgcacaag 3840 ctgcgcgctg taagcaacagcgctccgatc ggggtcaggc gtcgctctcg gtcccgcata 3900 tcgccatgga tcccgccgtctcccccgcga gcaccgaccc cctagatacc cacgcgtcgg 3960 gggccggggc ggccccgattccggtgtgcc ccacccccga gcggtacttc tacacctccc 4020 agtgccccga catcaaccaccttcgctccc tcagcatcct gaaccgctgg ctggagaccg 4080 agctcgtgtt cgtgggggacgaggaggacg tctccaagct ctccgagggc gagctcggct 4140 tctaccgctt tctgtttgccttcctgtcgg ccgcggacga cctggtgacg gaaaacctgg 4200 gcggcctctc cggcctcttcgaacagaagg acattcttca ctactacgtg gagcaggaat 4260 gcatcgaggt cgtccactcgcgcgtctaca acatcatcca gctggtgctc tttcacaaca 4320 acgaccaggc gcgccgcgcctatgtggccc gcaccatcaa ccacccggcc attcgcgtca 4380 aggtggactg gctggaggcgcgggtgcggg aatgcgactc gatcccggag aagttcatcc 4440 tcatgatcct catcgagggcgtcttttttg ccgcctcgtt cgccgccatc gcgtacctgc 4500 gcaccaacaa cctcctgcgggtcacctgcc agtcgaacga cctcatcagc cgcgacgagg 4560 ccgtgcatac gacagcctcgtgctacatct acaacaacta cctcgggggc cacgccaagc 4620 ccgaggcggc gcgcgtgtaccggctgtttc gggaggcggt ggatatcgag atcgggttca 4680 tccgatccca ggccccgacggacagctcta tcctgagtcc gggggccctg gcggccatcg 4740 agaactacgt gcgattcagcgcggatcgcc tgctgggcct gatccatatg cagcccctgt 4800 attccgcccc cgcccccgacgccagctttc ccctcagcct catgtccacc gacaaacaca 4860 ccaacttctt cgagtgccgcagcacctcgt acgccggggc cgtcgtcaac gatctgtgag 4920 ggtctgggcg cccttgtagcgatgtctaac cgaaataaag gggtcgaaac ggactgttgg 4980 gtctccggtg tgattattacgcaggggagg ggggtggcgg ctggggaaag ggaaggaacg 5040 cccgaaacca gagaaaaggaccaaaaggga aacgcgtcca accgataaat caagcgccga 5100 ccagaacccc gagatgcataataacaaacg attttattac tcttattatt aacaggtcgg 5160 gcatcgggag gggatgggggcgcgcgtttc ctccgttccg gctactcgtc ccagaattta 5220 gccaggacgt ccttgtaaaacgcgggcggg ggcgcgtggg cccacagctg cgccagaaac 5280 cggtcggcga tgtccggggcggtgatatgc cgagtcacga tggagcgcgc taaatcttcg 5340 tcgcggaggt cctgatagatgggcagtctt tttagaagag tccagggtcc ccgctccttg 5400 gggctgataa gcgatatgacgtacttgacg tatctgtgct ccaccagctc ggcgatggtc 5460 atcggatcgg gcagccagtccagggcctcc ggggcgtcgt ggatgacgtg gcggcgacgt 5520 ccggcgacat agccgcggtgttccgcgacc cgctgcgcgt tggggacctg cacgagctcg 5580 ggcggggtga gtatctccgaggaggacgac cgggcgccgt cgcgcggccc accggcgacg 5640 tccgggggct ggaggggggggtcttcttcg tagtcgtcct cgcccgcgat ctgttgggcc 5700 agaatttcgg tccacgagatgcgcgtctcg aggccgaccg gggccgcggt cagcgtaggc 5760 atgctctcca gggagcgcgagttggcgcgc tcccgccggg ccgcccggcg ggcctgggat 5820 cggctcgggg cggtccagtgacactcgcgc agcacgtcct cgacggacgc gtaggtgtta 5880 ttggggtgca ggtctgtgtggcagcggacg aacagcgcca ggaactgcgg gtaactcatc 5940 ttgaagtacc ctgcag 59562 1144 PRT herpes simplex virus type 2 2 Met Ala Asn Arg Pro Ala Ala SerAla Leu Ala Gly Ala Arg Ser Pro 1 5 10 15 Ser Glu Arg Gln Glu Pro ArgGlu Pro Glu Val Ala Pro Pro Gly Gly 20 25 30 Asp His Val Phe Cys Arg LysVal Ser Gly Val Met Val Leu Ser Ser 35 40 45 Asp Pro Pro Gly Pro Ala AlaTyr Arg Ile Ser Asp Ser Ser Phe Val 50 55 60 Gln Cys Gly Ser Asn Cys SerMet Ile Ile Asp Gly Asp Val Ala Arg 65 70 75 80 Gly His Leu Arg Asp LeuGlu Gly Ala Thr Ser Thr Gly Ala Phe Val 85 90 95 Ala Ile Ser Asn Val AlaAla Gly Gly Asp Gly Arg Thr Ala Val Val 100 105 110 Ala Leu Gly Gly ThrSer Gly Pro Ser Ala Thr Thr Ser Val Gly Thr 115 120 125 Gln Thr Ser GlyGlu Phe Leu His Gly Asn Pro Arg Thr Pro Glu Pro 130 135 140 Gln Gly ProGln Ala Val Pro Pro Pro Pro Pro Pro Pro Phe Pro Trp 145 150 155 160 GlyHis Glu Cys Cys Ala Arg Arg Asp Ala Arg Gly Gly Ala Glu Lys 165 170 175Asp Val Gly Ala Ala Glu Ser Trp Ser Asp Gly Pro Ser Ser Asp Ser 180 185190 Glu Thr Glu Asp Ser Asp Ser Ser Asp Glu Asp Thr Gly Ser Gly Ser 195200 205 Glu Thr Leu Ser Arg Ser Ser Ser Ile Trp Ala Ala Gly Ala Thr Asp210 215 220 Asp Asp Asp Ser Asp Ser Asp Ser Arg Ser Asp Asp Ser Val GlnPro 225 230 235 240 Asp Val Val Val Arg Arg Arg Trp Ser Asp Gly Pro AlaPro Val Ala 245 250 255 Phe Pro Lys Pro Arg Arg Pro Gly Asp Ser Pro GlyAsn Pro Gly Leu 260 265 270 Gly Ala Gly Thr Gly Pro Gly Ser Ala Thr AspPro Arg Ala Ser Ala 275 280 285 Asp Ser Asp Ser Ala Ala His Ala Ala AlaPro Gln Ala Asp Val Ala 290 295 300 Pro Val Leu Asp Ser Gln Pro Thr ValGly Thr Asp Pro Gly Tyr Pro 305 310 315 320 Val Pro Leu Glu Leu Thr ProGlu Asn Ala Glu Ala Val Ala Arg Phe 325 330 335 Leu Gly Asp Ala Val AspArg Glu Pro Ala Leu Met Leu Glu Tyr Phe 340 345 350 Cys Arg Cys Ala ArgGlu Glu Ser Lys Arg Val Pro Pro Arg Thr Phe 355 360 365 Gly Ser Ala ProArg Leu Thr Glu Asp Asp Phe Gly Leu Leu Asn Tyr 370 375 380 Ala Leu AlaGlu Met Arg Arg Leu Cys Leu Asp Leu Pro Pro Val Pro 385 390 395 400 ProAsn Ala Tyr Thr Pro Tyr His Leu Arg Glu Tyr Ala Thr Arg Leu 405 410 415Val Asn Gly Phe Lys Pro Leu Val Arg Arg Ser Ala Arg Leu Tyr Arg 420 425430 Ile Leu Gly Ile Leu Val His Leu Arg Ile Arg Thr Arg Glu Ala Ser 435440 445 Phe Glu Glu Trp Met Arg Ser Lys Glu Val Asp Leu Asp Phe Gly Leu450 455 460 Thr Glu Arg Leu Arg Glu His Glu Ala Gln Leu Met Ile Leu AlaGln 465 470 475 480 Ala Leu Asn Pro Tyr Asp Cys Leu Ile His Ser Thr ProAsn Thr Leu 485 490 495 Val Glu Arg Gly Leu Gln Ser Ala Leu Lys Tyr GluGlu Phe Tyr Leu 500 505 510 Lys Arg Phe Gly Gly His Tyr Met Glu Ser ValPhe Gln Met Tyr Thr 515 520 525 Arg Ile Ala Gly Phe Leu Ala Cys Arg AlaThr Arg Gly Met Arg His 530 535 540 Ile Ala Leu Gly Arg Gln Gly Ser TrpTrp Glu Met Phe Lys Phe Phe 545 550 555 560 Phe His Arg Leu Tyr Asp HisGln Ile Val Pro Ser Thr Pro Ala Met 565 570 575 Leu Asn Leu Gly Thr ArgAsn Tyr Tyr Thr Ser Ser Cys Tyr Leu Val 580 585 590 Asn Pro Gln Ala ThrThr Asn Gln Ala Thr Leu Arg Ala Ile Thr Gly 595 600 605 Asn Val Ser AlaIle Leu Ala Arg Asn Gly Gly Ile Gly Leu Cys Met 610 615 620 Gln Ala PheAsn Asp Ala Ser Pro Gly Thr Ala Ser Ile Met Pro Ala 625 630 635 640 LeuLys Val Leu Asp Ser Leu Val Ala Ala His Asn Lys Gln Ser Thr 645 650 655Arg Pro Thr Gly Ala Cys Val Tyr Leu Glu Pro Trp His Ser Asp Val 660 665670 Arg Ala Val Leu Arg Met Lys Gly Val Leu Ala Gly Glu Glu Ala Gln 675680 685 Arg Cys Asp Asn Ile Phe Ser Ala Leu Trp Met Pro Asp Leu Phe Phe690 695 700 Lys Arg Leu Ile Arg His Leu Asp Gly Glu Lys Asn Val Thr TrpSer 705 710 715 720 Leu Phe Asp Arg Asp Thr Ser Met Ser Leu Ala Asp PheHis Gly Glu 725 730 735 Glu Phe Glu Lys Leu Tyr Glu His Leu Glu Ala MetGly Phe Gly Glu 740 745 750 Thr Ile Pro Ile Gln Asp Leu Ala Tyr Ala IleVal Arg Ser Ala Ala 755 760 765 Thr Thr Gly Ser Pro Phe Ile Met Phe LysAsp Ala Val Asn Arg His 770 775 780 Tyr Ile Tyr Asp Thr Gln Gly Ala AlaIle Ala Gly Ser Asn Leu Cys 785 790 795 800 Thr Glu Ile Val His Pro SerSer Lys Arg Ser Ser Gly Val Cys Asn 805 810 815 Leu Gly Ser Val Asn LeuAla Arg Cys Val Ser Arg Arg Thr Phe Asp 820 825 830 Phe Gly Met Leu ArgAsp Ala Val Gln Ala Cys Val Leu Met Val Asn 835 840 845 Ile Met Ile AspSer Thr Leu Gln Pro Thr Pro Gln Cys Ala Arg Gly 850 855 860 His Asp AsnLeu Arg Ser Met Gly Ile Gly Met Gln Gly Leu His Thr 865 870 875 880 AlaCys Leu Lys Met Gly Leu Asp Leu Glu Ser Ala Glu Phe Arg Asp 885 890 895Leu Asn Thr His Ile Ala Glu Val Met Leu Leu Ala Ala Met Lys Thr 900 905910 Ser Asn Ala Leu Cys Val Arg Gly Ala Arg Pro Phe Ser His Phe Lys 915920 925 Arg Ser Met Tyr Arg Ala Gly Arg Phe His Trp Glu Arg Phe Ser Asn930 935 940 Ala Ser Pro Arg Tyr Glu Gly Glu Trp Glu Met Leu Arg Gln SerMet 945 950 955 960 Met Lys His Gly Leu Arg Asn Ser Gln Phe Ile Ala LeuMet Pro Thr 965 970 975 Ala Ala Ser Ala Gln Ile Ser Asp Val Ser Glu GlyPhe Ala Pro Leu 980 985 990 Phe Thr Asn Leu Phe Ser Lys Val Thr Arg AspGly Glu Thr Leu Arg 995 1000 1005 Pro Asn Thr Leu Leu Leu Lys Glu LeuGlu Arg Thr Phe Gly Gly Lys 1010 1015 1020 Arg Leu Leu Asp Ala Met AspGly Leu Glu Ala Lys Gln Trp Ser Val 1025 1030 1035 1040 Ala Gln Ala LeuPro Cys Leu Asp Pro Ala His Pro Leu Arg Arg Phe 1045 1050 1055 Lys ThrAla Phe Asp Tyr Asp Gln Glu Leu Leu Ile Asp Leu Cys Ala 1060 1065 1070Asp Arg Ala Pro Tyr Val Asp His Ser Gln Ser Met Thr Leu Tyr Val 10751080 1085 Thr Glu Lys Ala Asp Gly Thr Leu Pro Ala Ser Thr Leu Val ArgLeu 1090 1095 1100 Leu Val His Ala Tyr Lys Arg Gly Leu Lys Thr Gly MetTyr Tyr Cys 1105 1110 1115 1120 Lys Val Arg Lys Ala Thr Asn Ser Gly ValPhe Ala Gly Asp Asp Asn 1125 1130 1135 Ile Val Cys Thr Ser Cys Ala Leu1140

1. A method of inhibiting neuronal apoptosis n a mammal, said methodcomprising administering to said mammal an apoptosis-inhibiting amountof an isolated nucleic acid encoding ICP10, or any mutant, variant,homolog, or fragment thereof having anti-apoptotic activity.
 2. Themethod of claim 1, wherein said neuronal apoptosis is associated with aneurodegenerative disorder.
 3. The method of claim 2, wherein saidneurodegenerative disorder is selected from the group consisting ofAlzheimer's disease (AD), amyotropliic lateral sclerosis (ALS), Downsyndrome (DS), diabetic neuropathy, Parkinson's disease (PD), andHuntington disease (HD).
 4. The method of claim 1, wherein said neuronalapoptosis is associated with an injury of the central or peripheralnervous system.
 5. The method of claim 4, wherein said injury is theresult of stroke, cerebral ischemia, or chemical and/or physical trauma.6. A method of inhibiting neuronal apoptosis in a mammal, said methodcomprising administering to said mammal an apoptosis-inhibiting amountof the combination of an isolated nucleic acid encoding ICP10, or anymutant, variant, homolog, or fragment thereof having anti-apoptoticactivity, and an isolated nucleic acid encoding bcl-2, or any mutant,variant, homolog, or fragment thereof having anti-apoptotic activity. 7.The method of claim 6, wherein said isolated nucleic acid encoding ICP10and said isolated nucleic acid encoding bcl-2 are polycistronic.
 8. Amethod of inhibiting neuronal apoptosis in a mammal, said methodcomprising administering to said mammal an apoptosis-inhibiting amountof a vector comprising a nucleic acid encoding ICP10, or any mutant,variant, homolog, or fragment thereof having anti-apoptotic activity. 9.The method of claim 8, wherein said vector is selected from the groupconsisting of a virus and a plasmid.
 10. The method of claim 9, whereinsaid virus is selected from the group consisting of a herpesvirus,adenovirus, adeno associated virus, retrovirus, vaccinia virus, andcanary pox virus.
 11. The method of claim 10, wherein said herpesvirusis HSV-2.
 12. The method of claim 11, wherein HSV-2 comprises a mutationthat renders said HSV-2 replication-defective.
 13. The method of claim12, wherein said mutation eliminates the ribonucleotide reductase domainof ICP10.
 14. The method of claim 13, wherein said ribonucleotidereductase domain is replaced with nucleic acid selected from the groupconsisting of a nucleic acid encoding LacZ and a nucleic acid encodingbcl-2, or any mutant, variant, homolog, or fragment thereof havinganti-apoptotic activity.
 15. A method of inhibiting neuronal apoptosisin a mammal, said method comprising administering to said manual anapoptosis-inhibiting amount of a polypeptide encoded by a nucleic acidencoding ICP10, or any mutant, variant, homolog, or fragment thereofhaving anti-apoptotic activity.
 16. The method of claim 15, wherein saidpolypeptide is fused to a polypeptide encoded by a nucleic acid encodingbcl-2, or any mutant, variant, homolog, or fragment thereof havinganti-apoptotic activity.
 17. The method of claim 15, wherein saidneuronal apoptosis is associated with a neurodegenerative disorder. 18.The method of claim 17, wherein said neurodegenerative disorder isselected from the group consisting of Alzheimer's disease (AD),amyotrophic lateral sclerosis (ALS), Down syndrome (DS), diabeticneuropathy, Parkinson's disease (PD), and Huntington disease (HD). 19.The method of claim 15, wherein said neuronal apoptosis is associatedwith an injury of the central or peripheral nervous system.
 20. Themethod of claim 19, wherein said injury is the result of stroke,cerebral ischemia, or chemical and/or physical trauma.
 21. A method ofinhibiting neuronal apoptosis in a mammal, said method comprisingadministering to said mammal an apoptosis-inhibiting amount of thecombination of a polypeptide encoded by a nucleic acid encoding ICP10,or any mutant, variant, homolog, or fragment thereof having apoptoticactivity and a polypeptide encoded by a nucleic acid encoding bcl-2, orany mutant, variant, homolog, or fragment thereof having apoptoticactivity.
 22. A method of inhibiting apoptosis in a mammal, said methodcomprising administering to said mammal an apoptosis-inhibiting amountof an isolated nucleic acid encoding ICP10 or any mutant, variant,homolog, or fragment thereof having anti-apoptotic activity.
 23. Amethod of inhibiting apoptosis in a mammal, said method comprisingadministering to said mammal an apoptosis-inhibiting amount of thecombination of an isolated nucleic acid encoding ICP10, or any mutant,valiant, homolog, or fragment thereof having anti-apoptotic activity,and an isolated nucleic acid encoding bcl-2, or any mutant, variant,homolog, or fragment thereof having anti-apoptotic activity.
 24. Amethod of inhibiting apoptosis in a mammal, said method comprisingadministering to said mammal an apoptosis-inhibiting amount of a vectorcomprising a nucleic acid encoding ICP10, or any mutant, variant,homolog, or fragment thereof having anti-apoptotic activity.
 25. Amethod of inhibiting apoptosis in a mammal, said method comprisingadministering to said mammal an apoptosis-inhibiting amount of apolypeptide encoded by a nucleic acid encoding ICP10, or any mutant,variant, homolog, or fragment thereof having anti-apoptotic activity.26. A method of inhibiting apoptosis in a mammal, said method comprisingadministering to said mammal an apoptosis-inhibiting amount of apolypeptide encoded by a nucleic acid encoding ICP10, or any mutant,variant, homolog, or fragment thereof having anti-apoptotic activity.27. The method of claim 26, wherein said polypeptide is fused to apolypeptide encoded by a nucleic acid encoding bcl-2, or any mutant,variant, homolog, or fragment thereof having anti-apoptotic activity.28. A composition comprising a fusion polypeptide, wherein said fusionpolypeptide comprises a portion of ICP10 polypeptide and a portion of abcl-2 polypeptide, said composition further comprising apharmaceutically acceptable carrier therefor.